Sample records for mutated primer binding
-
Lapadat-Tapolsky, M; Gabus, C; Rau, M; Darlix, J L
1997-05-02
Retroviral nucleocapsid (NC) protein is an integral part of the virion nucleocapsid where it coats the dimeric RNA genome. Due to its nucleic acid binding and annealing activities, NC protein directs the annealing of the tRNA primer to the primer binding site and greatly facilitates minus strand DNA elongation and transfer while protecting the nucleic acids against nuclease degradation. To understand the role of NCp7 in viral DNA synthesis, we examined the influence of NCp7 on self-primed versus primer-specific reverse transcription. The results show that HIV-1 NCp7 can extensively inhibit self-primed reverse transcription of viral and cellular RNAs while promoting primer-specific synthesis of proviral DNA. The role of NCp7 vis-a-vis the presence of mutations in the viral DNA during minus strand elongation was examined. NCp7 maximized the annealing between a cDNA(-) primer containing one to five consecutive errors and an RNA representing the 3' end of the genome. The ability of reverse transcriptase (RT) in the presence of NCp7 to subsequently extend the mutated primers depended upon the position of the mismatch within the primer:template complex. When the mutations were at the polymerisation site, primer extension by RT in the presence of NCp7 was very high, about 40% for one mismatch and 3% for five consecutive mismatches. Mutations within the DNA primer or at its 5' end had little effect on the extension of viral DNA by RT. Taken together these results indicate that NCp7 plays major roles in proviral DNA synthesis within the virion core due to its ability to promote prime-specific proviral DNA synthesis while concurrently inhibiting non-specific reverse transcription of viral and cellular RNAs. Moreover, the observation that NCp7 enhances the incorporation of mutations during minus strand DNA elongation favours the notion that NCp7 is a factor contributing to the high mutation rate of HIV-1.
-
Neupauerová, Jana; Grečmalová, Dagmar; Seeman, Pavel; Laššuthová, Petra
2016-05-01
We describe a patient with early onset severe axonal Charcot-Marie-Tooth disease (CMT2) with dominant inheritance, in whom Sanger sequencing failed to detect a mutation in the mitofusin 2 (MFN2) gene because of a single nucleotide polymorphism (rs2236057) under the PCR primer sequence. The severe early onset phenotype and the family history with severely affected mother (died after delivery) was very suggestive of CMT2A and this suspicion was finally confirmed by a MFN2 mutation. The mutation p.His361Tyr was later detected in the patient by massively parallel sequencing with a gene panel for hereditary neuropathies. According to this information, new primers for amplification and sequencing were designed which bind away from the polymorphic sites of the patient's DNA. Sanger sequencing with these new primers then confirmed the heterozygous mutation in the MFN2 gene in this patient. This case report shows that massively parallel sequencing may in some rare cases be more sensitive than Sanger sequencing and highlights the importance of accurate primer design which requires special attention. © 2016 John Wiley & Sons Ltd/University College London.
-
Inokuchi, Shota; Yamashita, Yasuhiro; Nishimura, Kazuma; Nakanishi, Hiroaki; Saito, Kazuyuki
2017-11-01
Phenomena known as null alleles and peak imbalance can occur because of mutations in the primer binding sites used for DNA typing. In these cases, an accurate statistical evaluation of DNA typing is difficult. The estimated likelihood ratio is incorrectly calculated because of the null allele and allele dropout caused by mutation-induced peak imbalance. Although a number of studies have attempted to uncover examples of these phenomena, few reports are available on the human identification kit manufactured by Qiagen. In this study, 196 Japanese individuals who were heterozygous at D2S1360 were genotyped using an Investigator HDplex Kit with optimal amounts of DNA. A peak imbalance was frequently observed at the D2S1360 locus. We performed a sequencing analysis of the area surrounding the D2S1360 repeat motif to identify the cause for peak imbalance. A point mutation (G>A transition) 136 nucleotides upstream from the D2S1360 repeat motif was discovered in a number of samples. The allele frequency of the mutation was 0.0566 in the Japanese population. Therefore, human identification or kinship testing using the Investigator HDplex Kit requires caution because of the higher frequency of single nucleotide polymorphisms at the primer binding site of D2S1360 locus in the Japanese population.
-
Pflug, Alexander; Gaudon, Stephanie; Resa-Infante, Patricia; Lethier, Mathilde; Reich, Stefan; Schulze, Wiebke M
2018-01-01
Abstract Influenza polymerase uses short capped primers snatched from nascent Pol II transcripts to initiate transcription of viral mRNAs. Here we describe crystal structures of influenza A and B polymerase bound to a capped primer in a configuration consistent with transcription initiation (’priming state’) and show by functional assays that conserved residues from both the PB2 midlink and cap-binding domains are important for positioning the capped RNA. In particular, mutation of PB2 Arg264, which interacts with the triphosphate linkage in the cap, significantly and specifically decreases cap-dependent transcription. We also compare the configuration of the midlink and cap-binding domains in the priming state with their very different relative arrangement (called the ‘apo’ state) in structures where the potent cap-binding inhibitor VX-787, or a close analogue, is bound. In the ‘apo’ state the inhibitor makes additional interactions to the midlink domain that increases its affinity beyond that to the cap-binding domain alone. The comparison suggests that the mechanism of resistance of certain mutations that allow virus to escape from VX-787, notably PB2 N510T, can only be rationalized if VX-787 has a dual mode of action, direct inhibition of capped RNA binding as well as stabilization of the transcriptionally inactive ‘apo’ state. PMID:29202182
-
Yang, Young Geun; Kim, Jong Yeol; Park, Su Jeong; Kim, Suhng Wook; Jeon, Ok-Hee; Kim, Doo-Sik
2007-08-31
Apolipoprotein E (APOE) plays a critical role in lipoprotein metabolism by binding to both low-density lipoprotein and APOE receptors. The APOE gene has three allelic forms, epsilon2, epsilon3, and epsilon4, which encode different isoforms of the APOE protein. In this study, we have developed a new genotyping method for APOE. Our multiplex tetra-primer amplification refractory mutation system (multiplex T-ARMS) polymerase chain reaction (PCR) was performed in a single reaction tube with six primers consisting of two common primers and two specific primers for each of two single nucleotide polymorphism (SNP) sites. We obtained definitive electropherograms that showed three (epsilon2/epsilon2, epsilon3/epsilon3, and epsilon4/epsilon4), four (epsilon2/epsilon3 and epsilon3/epsilon4), and five (epsilon2/epsilon4) amplicons by multiplex T-ARMS PCR in a single reaction tube. Multiplex T-ARMS PCR for APOE genotyping is a simple and accurate method that requires only a single PCR reaction, without any another treatments or expensive instrumentation, to simultaneously identify two sites of single nucleotide polymorphisms.
-
Jones, Christopher P; Saadatmand, Jenan; Kleiman, Lawrence; Musier-Forsyth, Karin
2013-02-01
The primer for initiating reverse transcription in human immunodeficiency virus type 1 (HIV-1) is tRNA(Lys3). Host cell tRNA(Lys) is selectively packaged into HIV-1 through a specific interaction between the major tRNA(Lys)-binding protein, human lysyl-tRNA synthetase (hLysRS), and the viral proteins Gag and GagPol. Annealing of the tRNA primer onto the complementary primer-binding site (PBS) in viral RNA is mediated by the nucleocapsid domain of Gag. The mechanism by which tRNA(Lys3) is targeted to the PBS and released from hLysRS prior to annealing is unknown. Here, we show that hLysRS specifically binds to a tRNA anti-codon-like element (TLE) in the HIV-1 genome, which mimics the anti-codon loop of tRNA(Lys) and is located proximal to the PBS. Mutation of the U-rich sequence within the TLE attenuates binding of hLysRS in vitro and reduces the amount of annealed tRNA(Lys3) in virions. Thus, LysRS binds specifically to the TLE, which is part of a larger LysRS binding domain in the viral RNA that includes elements of the Psi packaging signal. Our results suggest that HIV-1 uses molecular mimicry of the anti-codon of tRNA(Lys) to increase the efficiency of tRNA(Lys3) annealing to viral RNA.
-
Jones, Christopher P.; Saadatmand, Jenan; Kleiman, Lawrence; Musier-Forsyth, Karin
2013-01-01
The primer for initiating reverse transcription in human immunodeficiency virus type 1 (HIV-1) is tRNALys3. Host cell tRNALys is selectively packaged into HIV-1 through a specific interaction between the major tRNALys-binding protein, human lysyl-tRNA synthetase (hLysRS), and the viral proteins Gag and GagPol. Annealing of the tRNA primer onto the complementary primer-binding site (PBS) in viral RNA is mediated by the nucleocapsid domain of Gag. The mechanism by which tRNALys3 is targeted to the PBS and released from hLysRS prior to annealing is unknown. Here, we show that hLysRS specifically binds to a tRNA anti-codon-like element (TLE) in the HIV-1 genome, which mimics the anti-codon loop of tRNALys and is located proximal to the PBS. Mutation of the U-rich sequence within the TLE attenuates binding of hLysRS in vitro and reduces the amount of annealed tRNALys3 in virions. Thus, LysRS binds specifically to the TLE, which is part of a larger LysRS binding domain in the viral RNA that includes elements of the Psi packaging signal. Our results suggest that HIV-1 uses molecular mimicry of the anti-codon of tRNALys to increase the efficiency of tRNALys3 annealing to viral RNA. PMID:23264568
-
A Nested-Splicing by Overlap Extension PCR Improves Specificity of this Standard Method.
Karkhane, Ali Asghar; Yakhchali, Bagher; Rastgar Jazii, Ferdous; Bambai, Bijan; Aminzadeh, Saeed; Rahimi, Fatemeh
2015-06-01
Splicing by overlap extension (SOE) PCR is used to create mutation in the coding sequence of an enzyme in order to study the role of specific residues in protein's structure and function. We introduced a nested-SOE-PCR (N -SOE-PCR) in order to increase the specificity and generating mutations in a gene by SOE-PCR. Genomic DNA from Bacillus thermocatenulatus was extracted. Nested PCR was used to amplify B. thermocatenulatus lipase gene variants, namely wild type and mutant, using gene specific and mutagenic specific primers, followed by cloning in a suitable vector. Briefly in N-SOE-PCR method, instead of two pairs of primers, three pairs of primers are used to amplify a mutagenic fragment. Moreover, the first and second PCR products are slightly longer than PCR products in a conventional SOE. PCR products obtained from the first round of PCR are used for the second PCR by applying the nested and mutated primers. Following to the purification of the amplified fragments, they will be subject of the further purification and will be used as template to perform the third round of PCR using gene specific primers. In the end, the products will be cloned into a suitable vector for subsequent application. In comparison to the conventional SOE-PCR, the improved method (i.e. N-SOE-PCR) increases the yield and specificity of the products. In addition, the proposed method shows a large reduction in the non-specific products. By applying two more primers in the conventional SOE, the specificity of the method will be improved. This would be in part due to annealing of the primers further inside the amplicon that increases both the efficiency and a better attachment of the primers. Positioning of the primer far from both ends of an amplicon leads to an enhanced binding as well as increased affinity in the third round of amplification in SOE.
-
Wan, Haisu; Li, Yongwen; Fan, Yu; Meng, Fanrong; Chen, Chen; Zhou, Qinghua
2012-01-15
Site-directed mutagenesis has become routine in molecular biology. However, many mutants can still be very difficult to create. Complicated chimerical mutations, tandem repeats, inverted sequences, GC-rich regions, and/or heavy secondary structures can cause inefficient or incorrect binding of the mutagenic primer to the target sequence and affect the subsequent amplification. In theory, these problems can be avoided by introducing the mutations into the target sequence using mutagenic fragments and so removing the need for primer-template annealing. The cassette mutagenesis uses the mutagenic fragment in its protocol; however, in most cases it needs to perform two rounds of mutagenic primer-based mutagenesis to introduce suitable restriction enzyme sites into templates and is not suitable for routine mutagenesis. Here we describe a highly efficient method in which the template except the region to be mutated is amplified by polymerase chain reaction (PCR) and the type IIs restriction enzyme-digested PCR product is directly ligated with the mutagenic fragment. Our method requires no assistance of mutagenic primers. We have used this method to create various types of difficult-to-make mutants with mutagenic frequencies of nearly 100%. Our protocol has many advantages over the prevalent QuikChange method and is a valuable tool for studies on gene structure and function. Copyright © 2011 Elsevier Inc. All rights reserved.
-
Yao, Yining; Yang, Qinrui; Shao, Chengchen; Liu, Baonian; Zhou, Yuxiang; Xu, Hongmei; Zhou, Yueqin; Tang, Qiqun; Xie, Jianhui
2018-01-01
Rare variants are widely observed in human genome and sequence variations at primer binding sites might impair the process of PCR amplification resulting in dropouts of alleles, named as null alleles. In this study, 5 cases from routine paternity testing using PowerPlex ® 21 System for STR genotyping were considered to harbor null alleles at TH01, FGA, D5S818, D8S1179, and D16S539, respectively. The dropout of alleles was confirmed by using alternative commercial kits AGCU Expressmarker 22 PCR amplification kit and AmpFℓSTR ® . Identifiler ® Plus Kit, and sequencing results revealed a single base variation at the primer binding site of each STR locus. Results from the collection of previous reports show that null alleles at D5S818 were frequently observed in population detected by two PowerPlex ® typing systems and null alleles at D19S433 were mostly observed in Japanese population detected by two AmpFℓSTR™ typing systems. Furthermore, the most popular mutation type appeared the transition from C to T with G to A, which might have a potential relationship with DNA methylation. Altogether, these results can provide helpful information in forensic practice to the elimination of genotyping discrepancy and the development of primer sets. Copyright © 2017 Elsevier B.V. All rights reserved.
-
Mikkelsen, Jacob Giehm; Lund, Anders H.; Dybkær, Karen; Duch, Mogens; Pedersen, Finn Skou
1998-01-01
We have previously demonstrated recombinational rescue of primer binding site (PBS)-impaired Akv murine leukemia virus-based vectors involving initial priming on endogenous viral sequences and template switching during cDNA synthesis to obtain PBS complementarity in second-strand transfer of reverse transcription (Mikkelsen et al., J. Virol. 70:1439–1447, 1996). By use of the same forced recombination system, we have now found recombinant proviruses of different structures, suggesting that PBS knockout vectors may be rescued through initial priming on endogenous virus RNA, read-through of the mutated PBS during minus-strand synthesis, and subsequent second-strand transfer mediated by the R-U5 complementarity of the plus strand and the extended minus-strand DNA acceptor template. Mechanisms for R-U5-mediated second-strand transfer and its possible role in retrovirus replication and evolution are discussed. PMID:9499117
-
Bartels, Stephan; Schipper, Elisa; Hasemeier, Britta; Kreipe, Hans; Lehmann, Ulrich
2018-05-27
The detection of hotspot mutations in key cancer genes is now an essential part of the diagnostic work-up in molecular pathology. Nearly all assays for mutation detection involve an amplification step. A second single nucleotide variant (SNV) on the same allele adjacent to a mutational hotspot can interfere with primer binding, leading to unnoticed allele-specific amplification of the wild type allele and thereby false-negative mutation testing. We present two diagnostic cases with false negative sequence results for JAK2 and SRSF2. In both cases mutations would have escaped detection if only one strand of DNA had been analysed. Because many commercially available diagnostic kits rely on the analysis of only one DNA strand they are prone to fail in cases like these. Detailed protocols and quality control measures to prevent corresponding pitfalls are presented. Copyright © 2017. Published by Elsevier Inc.
-
Genotyping of beta thalassemia trait by high-resolution DNA melting analysis.
Saetung, Rattika; Ongchai, Siriwan; Charoenkwan, Pimlak; Sanguansermsri, Torpong
2013-11-01
Beta thalassemia is a common hereditary hemalogogical disease in Thailand, with a prevalence of 5-8%. In this study, we evaluated the high resolution DNA melting (HRM) assay to identify beta thalassemia mutation in samples from 143 carriers of the beta thalassemia traits in at risk couples. The DNA was isolated from venous blood samples and tested for mutation under a series of 5 PCR-HRM (A, B, C, D and E primers) protocols. The A primers were for detection of beta thalassemia mutations in the HBB promoter region, the B primers for mutations in exon I, the C primers for exon II, the D primers for exon III and the E primers for the 3.4 kb deletion mutation. The mutations were diagnosed by comparing the complete melting curve profiles of a wild type control with those for each mutant sample. With the PCR-HRM technique, fourteen types of beta thalassemia mutations were detected. Each mutation had a unique and specific melting profile. The mutations included 36.4% (52 cases) codon 41/42-CTTT, 26.6% (38 cases) codon 17 A-T, 11.2% (16 cases) IVS1-1 G-T, 8.4% (12 cases) codon 71/72 +A, 8.4% (12 cases) of the 3.4 kb deletion and 3.5% (5 cases) -28 A-G. The remainder included one instance each of -87 C-A, -31 A-C, codon 27/28 +C, codon 30 G-A, IVS1-5 G-C, codon 35 C-A, codon 41-C and IVSII -654 C-T. Of the total cases, 85.8% of the mutations could be detected by primers B and C. The PCR-HRM method provides a rapid, simple and highly feasible strategy for mutation screening of beta thalassemia traits.
-
DOE Office of Scientific and Technical Information (OSTI.GOV)
Lishanski, A.; Ostrander, E.A.; Rine, J.
1994-03-29
An experimental strategy for detecting heterozygosity in genomic DNA has been developed based on preferential binding of Escherichia coli MutS protein to DNA molecules containing mismatched bases. The binding was detected by a gel mobility-shift assay. This approach was tested by using as a model the most commonly occurring mutations within the cystic fibrosis (CFTR) gene. Genomic DNA samples were amplified with 5{prime}-end-labeled primers that bracket the site of the {Delta}F508 3-bp deletion in exon 10 of the CFTR gene. The renatured PCR products from homozygotes produced homoduplexes; the PCR products from heterozygotes produced heteroduplexes and homoduplexes (1:1). MutS proteinmore » bound more strongly to heteroduplexes that correspond to heterozygous carriers of {Delta}F508 and contain a CTT or a GAA loop in one of the strands than to homoduplexes corresponding to homozygotes. The ability of MutS protein to detect heteroduplexes in PCR-amplified DNA extended to fragments {approximately} 500 bp long. The method was also able to detect carriers of the point mutations in exon 11 of the CFTR gene by a preferential binding of MutS to single-base mismatches in PCR-amplified DNA.« less
-
DOE Office of Scientific and Technical Information (OSTI.GOV)
Kim, Hee-Young; Kim, Hye-Young; Jung, Jaesung
2008-01-05
Our recent observation that hepatitis B virus (HBV) DNA polymerase (P) might initiate minus-strand DNA synthesis without primer [Kim et al., (2004) Virology 322, 22-30], raised a possibility that HBV P protein may have the potential to function as an RNA polymerase. Thus, we mutated Phe 436, a bulky amino acid with aromatic side chain, at the putative dNTP-binding cleft in reverse transcriptase (RT) domain of P protein to smaller amino acids (Gly or Val), and examined RNA polymerase activity. HBV core particles containing RT dNTP-binding cleft mutant P protein were able to incorporate {sup 32}P-ribonucleotides, but not HBV coremore » particles containing wild type (wt), priming-deficient mutant, or RT-deficient mutant P proteins. Since all the experiments were conducted with core particles isolated from transfected cells, our results indicate that the HBV RT mutant core particles containing RT dNTP-binding cleft mutant P protein could incorporate both deoxyribonucleotides and ribonucleotides in replicating systems.« less
-
DOE Office of Scientific and Technical Information (OSTI.GOV)
Richardson, C.C.
1993-12-31
This project focuses on the DNA polymerase (gene 5 protein) of phage T7 for use in DNA sequence analysis. Gene 5 protein interacts with accessory proteins to acquire properties essential for DNA replication. One goal is to understand these interactions in order to modify the proteins for use in DNA sequencing. E. coli thioredoxin, binds to gene 5 protein and clamps it to a primer-template. They have analyzed the binding of gene 5 protein-thioredoxin to primer-templates and have defined the optimal conditions to form an extremely stable complex with a dNTP in the polymerase catalytic site. The spatial proximity ofmore » these components has been determined using fluorescence emission anisotropy. The T7 DNA binding protein, the gene 2.5 protein, interacts with gene 5 protein and gene 4 protein to increase processivity and primer synthesis, respectively. Mutant gene 2.5 proteins have been isolated that do not interact with T7 DNA polymerase and can not support T7 growth. The nucleotide binding site of the T7 helicase has been identified and mutations affecting the site provide information on how the hydrolysis of NTPs fuel its unidirectional translocation. The sequence, GTC, has been shown to be necessary and sufficient for recognition by the T7 primase. The T7 gene 5.5 protein interacts with the E. coli nucleoid protein, H-NS, and also overcomes the phage {lambda} rex restriction system.« less
-
Kristensen, Lasse S; Andersen, Gitte B; Hager, Henrik; Hansen, Lise Lotte
2012-01-01
Sensitive and specific mutation detection is of particular importance in cancer diagnostics, prognostics, and individualized patient treatment. However, the majority of molecular methodologies that have been developed with the aim of increasing the sensitivity of mutation testing have drawbacks in terms of specificity, convenience, or costs. Here, we have established a new method, Competitive Amplification of Differentially Melting Amplicons (CADMA), which allows very sensitive and specific detection of all mutation types. The principle of the method is to amplify wild-type and mutated sequences simultaneously using a three-primer system. A mutation-specific primer is designed to introduce melting temperature decreasing mutations in the resulting mutated amplicon, while a second overlapping primer is designed to amplify both wild-type and mutated sequences. When combined with a third common primer very sensitive mutation detection becomes possible, when using high-resolution melting (HRM) as detection platform. The introduction of melting temperature decreasing mutations in the mutated amplicon also allows for further mutation enrichment by fast coamplification at lower denaturation temperature PCR (COLD-PCR). For proof-of-concept, we have designed CADMA assays for clinically relevant BRAF, EGFR, KRAS, and PIK3CA mutations, which are sensitive to, between 0.025% and 0.25%, mutated alleles in a wild-type background. In conclusion, CADMA enables highly sensitive and specific mutation detection by HRM analysis. © 2011 Wiley Periodicals, Inc.
-
Yang, Ji-Rong; Kuo, Chuan-Yi; Huang, Hsiang-Yi; Wu, Fu-Ting; Huang, Yi-Lung; Cheng, Chieh-Yu; Su, Yu-Ting; Chang, Feng-Yee; Wu, Ho-Sheng; Liu, Ming-Tsan
2014-01-01
New variants of the influenza A(H1N1)pdm09 and A(H3N2) viruses were detected in Taiwan between 2012 and 2013. Some of these variants were not detected in clinical specimens using a common real-time reverse transcription-PCR (RT-PCR) assay that targeted the conserved regions of the viral matrix (M) genes. An analysis of the M gene sequences of the new variants revealed that several newly emerging mutations were located in the regions where the primers or probes of the real-time RT-PCR assay bind; these included three mutations (G225A, T228C, and G238A) in the A(H1N1)pdm09 virus, as well as one mutation (C163T) in the A(H3N2) virus. These accumulated mismatch mutations, together with the previously identified C154T mutation of the A(H1N1)pdm09 virus and the C153T and G189T mutations of the A(H3N2) virus, result in a reduced detection sensitivity for the real-time RT-PCR assay. To overcome the loss of assay sensitivity due to mismatch mutations, we established a real-time RT-PCR assay using degenerate nucleotide bases in both the primers and probe and successfully increased the sensitivity of the assay to detect circulating variants of the human influenza A viruses. Our observations highlight the importance of the simultaneous use of different gene-targeting real-time RT-PCR assays for the clinical diagnosis of influenza.
-
DOE Office of Scientific and Technical Information (OSTI.GOV)
Song, Min-Suk; Kumar, Gyanendra; Shadrick, William R.
The influenza endonuclease is an essential subdomain of the viral RNA polymerase. It processes host pre-mRNAs to serve as primers for viral mRNA and is an attractive target for antiinfluenza drug discovery. Compound L-742,001 is a prototypical endonuclease inhibitor, and we found that repeated passaging of influenza virus in the presence of this drug did not lead to the development of resistant mutant strains. Reduced sensitivity to L-742,001 could only be induced by creating point mutations via a random mutagenesis strategy. Furthermore, these mutations mapped to the endonuclease active site where they can directly impact inhibitor binding. Engineered viruses containingmore » the mutations showed resistance to L-742,001 both in vitro and in vivo, with only a modest reduction in fitness. Introduction of the mutations into a second virus also increased its resistance to the inhibitor. When using the isolated wild-type and mutant endonuclease domains, we used kinetics, inhibitor binding and crystallography to characterize how the two most significant mutations elicit resistance to L-742,001. These studies lay the foundation for the development of a new class of influenza therapeutics with reduced potential for the development of clinical endonuclease inhibitor-resistant influenza strains.« less
-
Song, Min-Suk; Kumar, Gyanendra; Shadrick, William R.; ...
2016-03-14
The influenza endonuclease is an essential subdomain of the viral RNA polymerase. It processes host pre-mRNAs to serve as primers for viral mRNA and is an attractive target for antiinfluenza drug discovery. Compound L-742,001 is a prototypical endonuclease inhibitor, and we found that repeated passaging of influenza virus in the presence of this drug did not lead to the development of resistant mutant strains. Reduced sensitivity to L-742,001 could only be induced by creating point mutations via a random mutagenesis strategy. Furthermore, these mutations mapped to the endonuclease active site where they can directly impact inhibitor binding. Engineered viruses containingmore » the mutations showed resistance to L-742,001 both in vitro and in vivo, with only a modest reduction in fitness. Introduction of the mutations into a second virus also increased its resistance to the inhibitor. When using the isolated wild-type and mutant endonuclease domains, we used kinetics, inhibitor binding and crystallography to characterize how the two most significant mutations elicit resistance to L-742,001. These studies lay the foundation for the development of a new class of influenza therapeutics with reduced potential for the development of clinical endonuclease inhibitor-resistant influenza strains.« less
-
A Tale of Tails: Dissecting the Enhancing Effect of Tailed Primers in Real-Time PCR
Vandenbussche, Frank; Mathijs, Elisabeth; Lefebvre, David; De Clercq, Kris; Van Borm, Steven
2016-01-01
Non-specific tail sequences are often added to the 5’-terminus of primers to improve the robustness and overall performance of diagnostic assays. Despite the widespread use of tailed primers, the underlying working mechanism is not well understood. To address this problem, we conducted a detailed in vitro and in silico analysis of the enhancing effect of primer tailing on 2 well-established foot-and-mouth disease virus (FMDV) RT-qPCR assays using an FMDV reference panel. Tailing of the panFMDV-5UTR primers mainly affected the shape of the amplification curves. Modelling of the raw fluorescence data suggested a reduction of the amplification efficiency due to the accumulation of inhibitors. In depth analysis of PCR products indeed revealed the rapid accumulation of forward-primer derived artefacts. More importantly, tailing of the forward primer delayed artefacts formation and concomitantly restored the sigmoidal shape of the amplification curves. Our analysis also showed that primer tailing can alter utilisation patterns of degenerate primers and increase the number of primer variants that are able to participate in the reaction. The impact of tailed primers was less pronounced in the panFMDV-3D assay with only 5 out of 50 isolates showing a clear shift in Cq values. Sequence analysis of the target region of these 5 isolates revealed several mutations in the inter-primer region that extend an existing hairpin structure immediately downstream of the forward primer binding site. Stabilisation of the forward primer with either a tail sequence or cationic spermine units restored the sensitivity of the assay, which suggests that the enhancing effect in the panFMDV-3D assay is due to a more efficient extension of the forward primer. ur results show that primer tailing can alter amplification through various mechanisms that are determined by both the assay and target region. These findings expand our understanding of primer tailing and should enable a more targeted and efficient use of tailed primers. PMID:27723800
-
WASP: a Web-based Allele-Specific PCR assay designing tool for detecting SNPs and mutations
Wangkumhang, Pongsakorn; Chaichoompu, Kridsadakorn; Ngamphiw, Chumpol; Ruangrit, Uttapong; Chanprasert, Juntima; Assawamakin, Anunchai; Tongsima, Sissades
2007-01-01
Background Allele-specific (AS) Polymerase Chain Reaction is a convenient and inexpensive method for genotyping Single Nucleotide Polymorphisms (SNPs) and mutations. It is applied in many recent studies including population genetics, molecular genetics and pharmacogenomics. Using known AS primer design tools to create primers leads to cumbersome process to inexperience users since information about SNP/mutation must be acquired from public databases prior to the design. Furthermore, most of these tools do not offer the mismatch enhancement to designed primers. The available web applications do not provide user-friendly graphical input interface and intuitive visualization of their primer results. Results This work presents a web-based AS primer design application called WASP. This tool can efficiently design AS primers for human SNPs as well as mutations. To assist scientists with collecting necessary information about target polymorphisms, this tool provides a local SNP database containing over 10 million SNPs of various populations from public domain databases, namely NCBI dbSNP, HapMap and JSNP respectively. This database is tightly integrated with the tool so that users can perform the design for existing SNPs without going off the site. To guarantee specificity of AS primers, the proposed system incorporates a primer specificity enhancement technique widely used in experiment protocol. In particular, WASP makes use of different destabilizing effects by introducing one deliberate 'mismatch' at the penultimate (second to last of the 3'-end) base of AS primers to improve the resulting AS primers. Furthermore, WASP offers graphical user interface through scalable vector graphic (SVG) draw that allow users to select SNPs and graphically visualize designed primers and their conditions. Conclusion WASP offers a tool for designing AS primers for both SNPs and mutations. By integrating the database for known SNPs (using gene ID or rs number), this tool facilitates the awkward process of getting flanking sequences and other related information from public SNP databases. It takes into account the underlying destabilizing effect to ensure the effectiveness of designed primers. With user-friendly SVG interface, WASP intuitively presents resulting designed primers, which assist users to export or to make further adjustment to the design. This software can be freely accessed at . PMID:17697334
-
Zhang, Yunqing; Zhang, Xinju; Xu, Xiao; Kang, Zhihua; Li, Shibao; Zhang, Chen; Su, Bing
2014-01-01
A multiplex snapback primer system was developed for the simultaneous detection of JAK2 V617F and MPL W515L/K mutations in Philadelphia chromosome- (Ph-) negative myeloproliferative neoplasms (MPNs). The multiplex system comprises two snapback versus limiting primer sets for JAK2 and MPL mutation enrichment and detection, respectively. Linear-After exponential (LATE) PCR strategy was employed for the primer design to maximize the amplification efficiency of the system. Low ionic strength buffer and rapid PCR protocol allowed for selective amplification of the mutant alleles. Amplification products were analyzed by melting curve analysis for mutation identification. The multiplex system archived 0.1% mutation load sensitivity and <5% coefficient of variation inter-/intra-assay reproducibility. 120 clinical samples were tested by the multiplex snapback primer assay, and verified with amplification refractory system (ARMS), quantitative PCR (qPCR) and Sanger sequencing method. The multiplex system, with a favored versatility, provided the molecular diagnosis of Ph-negative MPNs with a suitable implement and simplified the genetic test process. PMID:24729973
-
Chen, Qianqian; Chen, Xiaoxiang; Zhang, Sichao; Lan, Ke; Lu, Jian; Zhang, Chiyu
2015-01-01
The development of simple, accurate, rapid and cost-effective technologies for mutation detection is crucial to the early diagnosis and prevention of numerous genetic diseases, pharmacogenetics, and drug resistance. Proofreading PCR (PR-PCR) was developed for mutation detection in 1998 but is rarely applied due to its low efficiency in allele discrimination. Here we developed a modified PR-PCR method using a ddNTP-blocked primer and a mixture of DNA polymerases with and without the 3'-5' proofreading function. The ddNTP-blocked primer exhibited the best blocking efficiency to avoid nonspecific primer extension while the mixture of a tiny amount of high-fidelity DNA polymerase with a routine amount of Taq DNA polymerase provided the best discrimination and amplification effects. The modified PR-PCR method is quite capable of detecting various mutation types, including point mutations and insertions/deletions (indels), and allows discrimination amplification when the mismatch is located within the last eight nucleotides from the 3'-end of the ddNTP-blocked primer. The modified PR-PCR has a sensitivity of 1-5 × 102 copies and a selectivity of 5 × 10-5 mutant among 107 copies of wild-type DNA. It showed a 100% accuracy rate in the detection of P72R germ-line mutation in the TP53 gene among 60 clinical blood samples, and a high potential to detect rifampin-resistant mutations at low frequency in Mycobacterium tuberculosis using an adaptor and a fusion-blocked primer. These results suggest that the modified PR-PCR technique is effective in detection of various mutations or polymorphisms as a simple, sensitive and promising approach. PMID:25915410
-
2013-01-01
Background Over the past decades site-directed mutagenesis (SDM) has become an indispensable tool for biological structure-function studies. In principle, SDM uses modified primer pairs in a PCR reaction to introduce a mutation in a cDNA insert. DpnI digestion of the reaction mixture is used to eliminate template copies before amplification in E. coli; however, this process is inefficient resulting in un-mutated clones which can only be distinguished from mutant clones by sequencing. Results We have developed a program – ‘SDM-Assist’ which creates SDM primers adding a specific identifier: through additional silent mutations a restriction site is included or a previous one removed which allows for highly efficient identification of ‘mutated clones’ by a simple restriction digest. Conclusions The direct identification of SDM clones will save time and money for researchers. SDM-Assist also scores the primers based on factors such as Tm, GC content and secondary structure allowing for simplified selection of optimal primer pairs. PMID:23522286
-
Ke, N; Gao, X; Keeney, J B; Boeke, J D; Voytas, D F
1999-01-01
Retrotransposons and retroviruses replicate by reverse transcription of an mRNA intermediate. Most retroelements initiate reverse transcription from a host-encoded tRNA primer. DNA synthesis typically extends from the 3'-OH of the acceptor stem, which is complementary to sequences on the retroelement mRNA (the primer binding site, PBS). However, for some retrotransposons, including the yeast Ty5 elements, sequences in the anticodon stem-loop of the initiator methionine tRNA (IMT) are complementary to the PBS. We took advantage of the genetic tractability of the yeast system to investigate the mechanism of Ty5 priming. We found that transposition frequencies decreased at least 800-fold for mutations in the Ty5 PBS that disrupt complementarity with the IMT. Similarly, transposition was reduced at least 200-fold for IMT mutations in the anticodon stem-loop. Base pairing between the Ty5 PBS and IMT is essential for transposition, as compensatory changes that restored base pairing between the two mutant RNAs restored transposition significantly. An analysis of 12 imt mutants with base changes outside of the region of complementarity failed to identify other tRNA residues important for transposition. In addition, assays carried out with heterologous IMTs from Schizosaccharomyces pombe and Arabidopsis thaliana indicated that residues outside of the anticodon stem-loop have at most a fivefold effect on transposition. Our genetic system should make it possible to further define the components required for priming and to understand the mechanism by which Ty5's novel primer is generated. PMID:10411136
-
Asari, Masaru; Okuda, Katsuhiro; Hoshina, Chisato; Omura, Tomohiro; Tasaki, Yoshikazu; Shiono, Hiroshi; Matsubara, Kazuo; Shimizu, Keiko
2016-02-01
The aim of this study was to develop a cost-effective genotyping method using high-quality DNA for human identification. A total of 21 short tandem repeats (STRs) and amelogenin were selected, and fluorescent fragments at 22 loci were simultaneously amplified in a single-tube reaction using locus-specific primers with 24-base universal tails and four fluorescent universal primers. Several nucleotide substitutions in universal tails and fluorescent universal primers enabled the detection of specific fluorescent fragments from the 22 loci. Multiplex polymerase chain reaction (PCR) produced intense FAM-, VIC-, NED-, and PET-labeled fragments ranging from 90 to 400 bp, and these fragments were discriminated using standard capillary electrophoretic analysis. The selected 22 loci were also analyzed using two commercial kits (the AmpFLSTR Identifiler Kit and the PowerPlex ESX 17 System), and results for two loci (D19S433 and D16S539) were discordant between these kits due to mutations at the primer binding sites. All genotypes from the 100 samples were determined using 2.5 ng of DNA by our method, and the expected alleles were completely recovered. Multiplex 22-locus genotyping using four fluorescent universal primers effectively reduces the costs to less than 20% of genotyping using commercial kits, and our method would be useful to detect silent alleles from commercial kit analysis. Copyright © 2015 Elsevier Inc. All rights reserved.
-
A multiplex method for detection of glucose-6-phosphate dehydrogenase (G6PD) gene mutations.
Zhang, L; Yang, Y; Liu, R; Li, Q; Yang, F; Ma, L; Liu, H; Chen, X; Yang, Z; Cui, L; He, Y
2015-12-01
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme defect caused by G6PD gene mutations. This study aimed to develop a cost-effective, multiplex, genotyping method for detecting common mutations in the G6PD gene. We used a SNaPshot approach to genotype multiple G6PD mutations that are common to human populations in South-East Asia. This assay is based on multiplex PCR coupled with primer extension reactions. Different G6PD gene mutations were determined by peak retention time and colors of the primer extension products. We designed PCR primers for multiplex amplification of the G6PD gene fragments and for primer extension reactions to genotype 11 G6PD mutations. DNA samples from a total of 120 unrelated G6PD-deficient individuals from the China-Myanmar border area were used to establish and validate this method. Direct sequencing of the PCR products demonstrated 100% concordance between the SNaPshot and the sequencing results. The SNaPshot method offers a specific and sensitive alternative for simultaneously interrogating multiple G6PD mutations. © 2015 John Wiley & Sons Ltd.
-
Alpha-tubulin missense mutations correlate with antimicrotubule drug resistance in Eleusine indica.
Yamamoto, E; Zeng, L; Baird, W V
1998-01-01
Dinitroaniline herbicides are antimicrotubule drugs that bind to tubulins and inhibit polymerization. As a result of repeated application of dinitroaniline herbicides, highly resistant and intermediately resistant biotypes of goosegrass (Eleusine indica) developed in previously wild-type populations. Three alpha-tubulin cDNA classes (designated TUA1, TUA2, and TUA3) were isolated from each biotype. Nucleotide differences between the susceptible and the resistant (R) alpha-tubulins were identified in TUA1 and TUA2. The most significant differences were missense mutations that occurred in TUA1 of the R and intermediately resistant (I) biotypes. Such mutations convert Thr-239 to Ile in the R biotype and Met-268 to Thr in the I biotype. These amino acid substitutions alter hydrophobicity; therefore, they may alter the dinitroaniline binding property of the protein. These mutations were correlated with the dinitroaniline response phenotypes (Drp). Plants homozygous for susceptibility possessed the wild-type TUA1 allele; plants homozygous for resistance possessed the mutant tua1 allele; and plants heterozygous for susceptibility possessed both wild-type and mutant alleles. Thus, we conclude that TUA1 is at the Drp locus. Using polymerase chain reaction primer-introduced restriction analysis, we demonstrated that goosegrass genomic DNA can be diagnosed for Drp alleles. Although not direct proof, these results suggest that a mutation in an alpha-tubulin gene confers resistance to dinitroanilines in goosegrass. PMID:9490751
-
Alpha-tubulin missense mutations correlate with antimicrotubule drug resistance in Eleusine indica.
Yamamoto, E; Zeng, L; Baird, W V
1998-02-01
Dinitroaniline herbicides are antimicrotubule drugs that bind to tubulins and inhibit polymerization. As a result of repeated application of dinitroaniline herbicides, highly resistant and intermediately resistant biotypes of goosegrass (Eleusine indica) developed in previously wild-type populations. Three alpha-tubulin cDNA classes (designated TUA1, TUA2, and TUA3) were isolated from each biotype. Nucleotide differences between the susceptible and the resistant (R) alpha-tubulins were identified in TUA1 and TUA2. The most significant differences were missense mutations that occurred in TUA1 of the R and intermediately resistant (I) biotypes. Such mutations convert Thr-239 to Ile in the R biotype and Met-268 to Thr in the I biotype. These amino acid substitutions alter hydrophobicity; therefore, they may alter the dinitroaniline binding property of the protein. These mutations were correlated with the dinitroaniline response phenotypes (Drp). Plants homozygous for susceptibility possessed the wild-type TUA1 allele; plants homozygous for resistance possessed the mutant tua1 allele; and plants heterozygous for susceptibility possessed both wild-type and mutant alleles. Thus, we conclude that TUA1 is at the Drp locus. Using polymerase chain reaction primer-introduced restriction analysis, we demonstrated that goosegrass genomic DNA can be diagnosed for Drp alleles. Although not direct proof, these results suggest that a mutation in an alpha-tubulin gene confers resistance to dinitroanilines in goosegrass.
-
French, Deborah; Smith, Andrew; Powers, Martin P; Wu, Alan H B
2011-08-17
Binding of a ligand to the epidermal growth factor receptor (EGFR) stimulates various intracellular signaling pathways resulting in cell cycle progression, proliferation, angiogenesis and apoptosis inhibition. KRAS is involved in signaling pathways including RAF/MAPK and PI3K and mutations in this gene result in constitutive activation of these pathways, independent of EGFR activation. Seven mutations in codons 12 and 13 of KRAS comprise around 95% of the observed human mutations, rendering monoclonal antibodies against EGFR (e.g. cetuximab and panitumumab) useless in treatment of colorectal cancer. KRAS mutation testing by two different methodologies was compared; Sanger sequencing and AutoGenomics INFINITI® assay, on DNA extracted from colorectal cancers. Out of 29 colorectal tumor samples tested, 28 were concordant between the two methodologies for the KRAS mutations that were detected in both assays with the INFINITI® assay detecting a mutation in one sample that was indeterminate by Sanger sequencing and a third methodology; single nucleotide primer extension. This study indicates the utility of the AutoGenomics INFINITI® methodology in a clinical laboratory setting where technical expertise or access to equipment for DNA sequencing does not exist. Copyright © 2011 Elsevier B.V. All rights reserved.
-
Fenstermacher, Katherine J; Achuthan, Vasudevan; Schneider, Thomas D; DeStefano, Jeffrey J
2018-01-16
DNA polymerases (DNAPs) recognize 3' recessed termini on duplex DNA and carry out nucleotide catalysis. Unlike promoter-specific RNA polymerases (RNAPs), no sequence specificity is required for binding or initiation of catalysis. Despite this, previous results indicate that viral reverse transcriptases bind much more tightly to DNA primers that mimic the polypurine tract. In the current report, primer sequences that bind with high affinity to Taq and Klenow polymerases were identified using a modified Selective Evolution of Ligands by Exponential Enrichment (SELEX) approach. Two Taq -specific primers that bound ∼10 (Taq1) and over 100 (Taq2) times more stably than controls to Taq were identified. Taq1 contained 8 nucleotides (5' -CACTAAAG-3') that matched the phage T3 RNAP "core" promoter. Both primers dramatically outcompeted primers with similar binding thermodynamics in PCR reactions. Similarly, exonuclease minus Klenow polymerase also selected a high affinity primer that contained a related core promoter sequence from phage T7 RNAP (5' -ACTATAG-3'). For both Taq and Klenow, even small modifications to the sequence resulted in large losses in binding affinity suggesting that binding was highly sequence-specific. The results are discussed in the context of possible effects on multi-primer (multiplex) PCR assays, molecular information theory, and the evolution of RNAPs and DNAPs. Importance This work further demonstrates that primer-dependent DNA polymerases can have strong sequence biases leading to dramatically tighter binding to specific sequences. These may be related to biological function, or be a consequences of the structural architecture of the enzyme. New sequence specificity for Taq and Klenow polymerases were uncovered and among them were sequences that contained the core promoter elements from T3 and T7 phage RNA polymerase promoters. This suggests the intriguing possibility that phage RNA polymerases exploited intrinsic binding affinities of ancestral DNA polymerases to develop their promotors. Conversely, DNA polymerases could have evolved from related RNA polymerases and retained the intrinsic binding preference despite there being no clear function for such a preference in DNA biology. Copyright © 2018 American Society for Microbiology.
-
Ahlawat, Sonika; Sharma, Rekha; Maitra, A.; Roy, Manoranjan; Tantia, M.S.
2014-01-01
New, quick, and inexpensive methods for genotyping novel caprine Fec gene polymorphisms through tetra-primer ARMS PCR were developed in the present investigation. Single nucleotide polymorphism (SNP) genotyping needs to be attempted to establish association between the identified mutations and traits of economic importance. In the current study, we have successfully genotyped three new SNPs identified in caprine fecundity genes viz. T(-242)C (BMPR1B), G1189A (GDF9) and G735A (BMP15). Tetra-primer ARMS PCR protocol was optimized and validated for these SNPs with short turn-around time and costs. The optimized techniques were tested on 158 random samples of Black Bengal goat breed. Samples with known genotypes for the described genes, previously tested in duplicate using the sequencing methods, were employed for validation of the assay. Upon validation, complete concordance was observed between the tetra-primer ARMS PCR assays and the sequencing results. These results highlight the ability of tetra-primer ARMS PCR in genotyping of mutations in Fec genes. Any associated SNP could be used to accelerate the improvement of goat reproductive traits by identifying high prolific animals at an early stage of life. Our results provide direct evidence that tetra-primer ARMS-PCR is a rapid, reliable, and cost-effective method for SNP genotyping of mutations in caprine Fec genes. PMID:25606428
-
A new rapid methodological strategy to assess BRCA mutational status.
Vuttariello, Emilia; Borra, Marco; Calise, Celeste; Mauriello, Elvira; Greggi, Stefano; Vecchione, Aldo; Biffali, Elio; Chiappetta, Gennaro
2013-07-01
Hereditary cancers account for approximately 10 % of breast and ovarian cancers. Mutations of the BRCA1 and BRCA2 genes, encoding two proteins involved in DNA repair, underlie most cases of such hereditary cancers. Women with BRCA mutations develop breast cancer in 50-80 % of cases and ovarian cancer in 10-40 % of cases. Assessing BRCA mutational status is needed to direct the clinical management of women with predisposition to these hereditary cancers. However, BRCA screening constitutes a bottleneck in terms of costs and time to deliver results. We developed a PCR-based assay using 73 primer pairs covering the entire coding regions of BRCA1 and BRCA2. PCR primers, containing at the 5' end the universal M13 primer sequences, were pre-spotted in 96-well plates. Following PCR, direct sequencing was performed using M13 primers, allowing to standardize the conditions. PCR amplification and sequencing were successful for each amplicon. We tested and validated the assay on 10 known gDNAs from patients with Hereditary breast and ovarian cancer (HBOC). Our strategy is a promising time and cost-effective method to detect BRCA mutations in the clinical setting, which is essential to formulate a personalized therapy for patients with HBOC.
-
Liang, Chen; Rong, Liwei; Russell, Rodney S.; Wainberg, Mark A.
2000-01-01
We have studied the role of an RNA region at nucleotides (nt) +200 to +233, just downstream of the 5′ long terminal repeat, in encapsidation of human immunodeficiency virus type 1 genomic RNA. Three deletion mutations, namely, BH-D0, BH-D1, and BH-D2, were generated to eliminate sequences at positions nt +200 to +219, +200 to +226, and +200 to +233. The result in each case was decreased levels of packaging of viral RNA into the mutated viruses, with the BH-D2 virus being the most severely affected. Consistently, all three deletions resulted in impaired viral infectiousness and the BH-D2 mutation showed the most dramatic impact in this regard. Further analysis revealed additional defects in Gag precursor processing and in the extension efficiency of the tRNA3Lys primer in reverse transcription reactions performed with these mutated viruses. To shed further light on the function of these deleted sequences in viral replication, the mutated viruses were cultured in MT-2 cells over prolonged periods to enable them to reacquire wild-type replication kinetics. Sequencing of the reverted viruses revealed point mutations in both the noncoding region and the gag gene. In the case of the BH-D0 revertant, two mutations were observed at positions G112A in the U5 region, termed M1, and T24I in the nucleocapsid protein, termed MNC, respectively. Either of these two mutations was able to confer wild-type replication capacity on BH-D0. In the case of BH-D1, each of the M1 mutations, a mutation termed M2, i.e., C227T, just downstream of the primer binding site, a mutation termed MP2 (T12I) in the p2 protein, and the MNC mutation were observed. A combination of either M1 and M2 or MP2 and MNC was able to rescue BH-D1. In the case of the BH-D2 deletion-containing viruses, three point mutations, i.e., M1, MP2, and MNC, were observed and the presence of all three was required to restore viral replication to wild-type levels. PMID:10864634
-
Distribution of mutations in the PEX gene in families with X-linked hypophosphataemic rickets (HYP).
Rowe, P S; Oudet, C L; Francis, F; Sinding, C; Pannetier, S; Econs, M J; Strom, T M; Meitinger, T; Garabedian, M; David, A; Macher, M A; Questiaux, E; Popowska, E; Pronicka, E; Read, A P; Mokrzycki, A; Glorieux, F H; Drezner, M K; Hanauer, A; Lehrach, H; Goulding, J N; O'Riordan, J L
1997-04-01
Mutations in the PEX gene at Xp22.1 (phosphate-regulating gene with homologies to endopeptidases, on the X-chromosome), are responsible for X-linked hypophosphataemic rickets (HYP). Homology of PEX to the M13 family of Zn2+ metallopeptidases which include neprilysin (NEP) as prototype, has raised important questions regarding PEX function at the molecular level. The aim of this study was to analyse 99 HYP families for PEX gene mutations, and to correlate predicted changes in the protein structure with Zn2+ metallopeptidase gene function. Primers flanking 22 characterised exons were used to amplify DNA by PCR, and SSCP was then used to screen for mutations. Deletions, insertions, nonsense mutations, stop codons and splice mutations occurred in 83% of families screened for in all 22 exons, and 51% of a separate set of families screened in 17 PEX gene exons. Missense mutations in four regions of the gene were informative regarding function, with one mutation in the Zn2+-binding site predicted to alter substrate enzyme interaction and catalysis. Computer analysis of the remaining mutations predicted changes in secondary structure, N-glycosylation, protein phosphorylation and catalytic site molecular structure. The wide range of mutations that align with regions required for protease activity in NEP suggests that PEX also functions as a protease, and may act by processing factor(s) involved in bone mineral metabolism.
-
Lin, J H; Levin, H L
1997-01-15
All retroviruses and LTR-containing retrotransposons are thought to require specific tRNA molecules to serve as primers of reverse transcription. An exception is the LTR-containing retrotransposon Tf1, isolated from Schizosaccharomyces pombe. Instead of requiring a tRNA, the reverse transcriptase of Tf1 uses the first 11 bases of the Tf1 transcript as the primer for reverse transcription. The primer is generated by a cleavage that occurs between bases 11 and 12 of the Tf1 mRNA. Sequence analysis of the 5' untranslated region of the Tf1 mRNA resulted in the identification of a region with the potential to form an RNA structure of 89 bases that included the primer binding site and the first 11 bases of the Tf1 mRNA. Systematic mutagenesis of this region revealed 34 single-point mutants in the structure that resulted in reduced transposition activity. The defects in transposition correlated with reduced level of Tf1 reverse transcripts as determined by DNA blot analysis. Evidence that the RNA structure did form in vivo included the result that strains with second site mutations that restored complementarity resulted in increased levels of reverse transcripts and Tf1 transposition. The majority of the mutants defective for reverse transcription were unable to cleave the Tf1 mRNA between bases 11 and 12. These data indicate that formation of an extensive RNA structure was required for the cleavage reaction that generated the primer for Tf1 reverse transcription.
-
Mingo, Janire; Erramuzpe, Asier; Luna, Sandra; Aurtenetxe, Olaia; Amo, Laura; Diez, Ibai; Schepens, Jan T. G.; Hendriks, Wiljan J. A. J.; Cortés, Jesús M.; Pulido, Rafael
2016-01-01
Site-directed mutagenesis (SDM) is a powerful tool to create defined collections of protein variants for experimental and clinical purposes, but effectiveness is compromised when a large number of mutations is required. We present here a one-tube-only standardized SDM approach that generates comprehensive collections of amino acid substitution variants, including scanning- and single site-multiple mutations. The approach combines unified mutagenic primer design with the mixing of multiple distinct primer pairs and/or plasmid templates to increase the yield of a single inverse-PCR mutagenesis reaction. Also, a user-friendly program for automatic design of standardized primers for Ala-scanning mutagenesis is made available. Experimental results were compared with a modeling approach together with stochastic simulation data. For single site-multiple mutagenesis purposes and for simultaneous mutagenesis in different plasmid backgrounds, combination of primer sets and/or plasmid templates in a single reaction tube yielded the distinct mutations in a stochastic fashion. For scanning mutagenesis, we found that a combination of overlapping primer sets in a single PCR reaction allowed the yield of different individual mutations, although this yield did not necessarily follow a stochastic trend. Double mutants were generated when the overlap of primer pairs was below 60%. Our results illustrate that one-tube-only SDM effectively reduces the number of reactions required in large-scale mutagenesis strategies, facilitating the generation of comprehensive collections of protein variants suitable for functional analysis. PMID:27548698
-
Fuchs, Ryan T.; Grundy, Frank J.; Henkin, Tina M.
2007-01-01
The SMK box is a conserved riboswitch motif found in the 5′ untranslated region of metK genes [encoding S-adenosylmethionine (SAM) synthetase] in lactic acid bacteria, including Enterococcus, Streptococcus, and Lactococcus sp. Previous studies showed that this RNA element binds SAM in vitro, and SAM binding causes a structural rearrangement that sequesters the Shine–Dalgarno (SD) sequence by pairing with an anti-SD (ASD) element. A model was proposed in which SAM binding inhibits metK translation by preventing binding of the ribosome to the SD region of the mRNA. In the current work, the addition of SAM was shown to inhibit binding of 30S ribosomal subunits to SMK box RNA; in contrast, the addition of S-adenosylhomocysteine (SAH) had no effect. A mutant RNA, which has a disrupted SD-ASD pairing, was defective in SAM binding and showed no reduction of ribosome binding in the presence of SAM, whereas a compensatory mutation that restored SD-ASD pairing restored the response to SAM. Primer extension inhibition assays provided further evidence for SD-ASD pairing in the presence of SAM. These results strongly support the model that SMK box translational repression operates through occlusion of the ribosome binding site and that SAM binding requires the SD-ASD pairing. PMID:17360376
-
van der Linden, Lonneke; Vives-Adrián, Laia; Selisko, Barbara; Ferrer-Orta, Cristina; Liu, Xinran; Lanke, Kjerstin; Ulferts, Rachel; De Palma, Armando M; Tanchis, Federica; Goris, Nesya; Lefebvre, David; De Clercq, Kris; Leyssen, Pieter; Lacroix, Céline; Pürstinger, Gerhard; Coutard, Bruno; Canard, Bruno; Boehr, David D; Arnold, Jamie J; Cameron, Craig E; Verdaguer, Nuria; Neyts, Johan; van Kuppeveld, Frank J M
2015-03-01
The genus Enterovirus of the family Picornaviridae contains many important human pathogens (e.g., poliovirus, coxsackievirus, rhinovirus, and enterovirus 71) for which no antiviral drugs are available. The viral RNA-dependent RNA polymerase is an attractive target for antiviral therapy. Nucleoside-based inhibitors have broad-spectrum activity but often exhibit off-target effects. Most non-nucleoside inhibitors (NNIs) target surface cavities, which are structurally more flexible than the nucleotide-binding pocket, and hence have a more narrow spectrum of activity and are more prone to resistance development. Here, we report a novel NNI, GPC-N114 (2,2'-[(4-chloro-1,2-phenylene)bis(oxy)]bis(5-nitro-benzonitrile)) with broad-spectrum activity against enteroviruses and cardioviruses (another genus in the picornavirus family). Surprisingly, coxsackievirus B3 (CVB3) and poliovirus displayed a high genetic barrier to resistance against GPC-N114. By contrast, EMCV, a cardiovirus, rapidly acquired resistance due to mutations in 3Dpol. In vitro polymerase activity assays showed that GPC-N114 i) inhibited the elongation activity of recombinant CVB3 and EMCV 3Dpol, (ii) had reduced activity against EMCV 3Dpol with the resistance mutations, and (iii) was most efficient in inhibiting 3Dpol when added before the RNA template-primer duplex. Elucidation of a crystal structure of the inhibitor bound to CVB3 3Dpol confirmed the RNA-binding channel as the target for GPC-N114. Docking studies of the compound into the crystal structures of the compound-resistant EMCV 3Dpol mutants suggested that the resistant phenotype is due to subtle changes that interfere with the binding of GPC-N114 but not of the RNA template-primer. In conclusion, this study presents the first NNI that targets the RNA template channel of the picornavirus polymerase and identifies a new pocket that can be used for the design of broad-spectrum inhibitors. Moreover, this study provides important new insight into the plasticity of picornavirus polymerases at the template binding site.
-
van der Linden, Lonneke; Vives-Adrián, Laia; Selisko, Barbara; Ferrer-Orta, Cristina; Liu, Xinran; Lanke, Kjerstin; Ulferts, Rachel; De Palma, Armando M.; Tanchis, Federica; Goris, Nesya; Lefebvre, David; De Clercq, Kris; Leyssen, Pieter; Lacroix, Céline; Pürstinger, Gerhard; Coutard, Bruno; Canard, Bruno; Boehr, David D.; Arnold, Jamie J.; Cameron, Craig E.; Verdaguer, Nuria
2015-01-01
The genus Enterovirus of the family Picornaviridae contains many important human pathogens (e.g., poliovirus, coxsackievirus, rhinovirus, and enterovirus 71) for which no antiviral drugs are available. The viral RNA-dependent RNA polymerase is an attractive target for antiviral therapy. Nucleoside-based inhibitors have broad-spectrum activity but often exhibit off-target effects. Most non-nucleoside inhibitors (NNIs) target surface cavities, which are structurally more flexible than the nucleotide-binding pocket, and hence have a more narrow spectrum of activity and are more prone to resistance development. Here, we report a novel NNI, GPC-N114 (2,2'-[(4-chloro-1,2-phenylene)bis(oxy)]bis(5-nitro-benzonitrile)) with broad-spectrum activity against enteroviruses and cardioviruses (another genus in the picornavirus family). Surprisingly, coxsackievirus B3 (CVB3) and poliovirus displayed a high genetic barrier to resistance against GPC-N114. By contrast, EMCV, a cardiovirus, rapidly acquired resistance due to mutations in 3Dpol. In vitro polymerase activity assays showed that GPC-N114 i) inhibited the elongation activity of recombinant CVB3 and EMCV 3Dpol, (ii) had reduced activity against EMCV 3Dpol with the resistance mutations, and (iii) was most efficient in inhibiting 3Dpol when added before the RNA template-primer duplex. Elucidation of a crystal structure of the inhibitor bound to CVB3 3Dpol confirmed the RNA-binding channel as the target for GPC-N114. Docking studies of the compound into the crystal structures of the compound-resistant EMCV 3Dpol mutants suggested that the resistant phenotype is due to subtle changes that interfere with the binding of GPC-N114 but not of the RNA template-primer. In conclusion, this study presents the first NNI that targets the RNA template channel of the picornavirus polymerase and identifies a new pocket that can be used for the design of broad-spectrum inhibitors. Moreover, this study provides important new insight into the plasticity of picornavirus polymerases at the template binding site. PMID:25799064
-
Hoy, M.S.; Kelly, K.; Rodriguez, R.J.
2010-01-01
A 3-primer PCR system was developed to discriminate invasive zebra (Dreissena polymorpha) and quagga (Dreissena bugensis) mussel. The system is based on: 1) universal primers that amplifies a region of the nuclear 28s rDNA gene from both species and 2) a species-specific primer complementary to either zebra or quagga mussel. The species-specific primers bind to sequences between the binding sites for the universal primers resulting in the amplification of two products from the target species and one product from the nontarget species. Therefore, nontarget products are positive amplification controls. The 3-primer system accurately discriminated zebra and quagga mussels from seven geographically distinct populations.
-
Liu, Dewu; Zhang, Yushan; Du, Yinjun; Yang, Guanfu; Zhang, Xiquan
2007-06-01
The growth-correlated genes that are part of the neuroendocrine growth axis play crucial roles in the regulation of growth and development of pig. The identification of genetic polymorphisms in these genes will enable the scientist to evaluate the biological relevance of such polymorphisms and to gain a better understanding of quantitative traits like growth. In the present study, seven pairs of primers were designed to obtain unknown sequences of growth-correlated genes, and other 25 pairs of primers were designed to identify single nucleotide polymorphisms (SNP) using the denaturing high-performance liquid chromatography (DHPLC) technology in four pig breeds (Duroc, Landrace, Lantang and Wuzhishan), significantly differing in growth and development characteristics. A total of 101 polymorphisms were discovered in 10,707 base pairs (bp) from six genes of the ghrelin (GHRL), leptin (LEP), insulin-like growth factor II (IGF-II), insulin-like growth factor binding protein 2 (IGFBP-2), insulin-like growth factor binding protein 3 (IGFBP-3), and somatostatin (SS). The observed average distances between the SNP in the 5'UTR, coding regions, introns and 3'UTR were 134, 521, 81 and 92 bp, respectively. Four SNPs were found in the coding regions of IGF-II, IGFBP-2 and LEP, respectively. Two synonymous mutations were obtained in IGF-II and LEP genes respectively, and two non-synonymous were found in IGFBP-2 and LEP genes, respectively. Seven other mutations were also observed. Thirty-two PCR-RFLP markers were found among 101 polymorphisms of the six genes. The SNP discovered in this study would provide suitable markers for association studies of candidate genes with growth related traits in pig.
-
NASA Astrophysics Data System (ADS)
Tsao, Shih-Ming; Lai, Ji-Ching; Horng, Horng-Er; Liu, Tu-Chen; Hong, Chin-Yih
2017-04-01
Aptamers are oligonucleotides that can bind to specific target molecules. Most aptamers are generated using random libraries in the standard systematic evolution of ligands by exponential enrichment (SELEX). Each random library contains oligonucleotides with a randomized central region and two fixed primer regions at both ends. The fixed primer regions are necessary for amplifying target-bound sequences by PCR. However, these extra-sequences may cause non-specific bindings, which potentially interfere with good binding for random sequences. The Magnetic-Assisted Rapid Aptamer Selection (MARAS) is a newly developed protocol for generating single-strand DNA aptamers. No repeat selection cycle is required in the protocol. This study proposes and demonstrates a method to isolate aptamers for C-reactive proteins (CRP) from a randomized ssDNA library containing no fixed sequences at 5‧ and 3‧ termini using the MARAS platform. Furthermore, the isolated primer-free aptamer was sequenced and binding affinity for CRP was analyzed. The specificity of the obtained aptamer was validated using blind serum samples. The result was consistent with monoclonal antibody-based nephelometry analysis, which indicated that a primer-free aptamer has high specificity toward targets. MARAS is a feasible platform for efficiently generating primer-free aptamers for clinical diagnoses.
-
Rapid polymerase chain reaction screening of Helicobacter pylori chromosomal point mutations.
Ge, Z; Taylor, D E
1997-09-01
Microdiversity (within individual genes) in the genomes of different Helicobacter pylori strains has been demonstrated to be more frequent than that seen in other prokaryotes. Point mutations in some genes, such as the vacA and 23S ribosomal RNA genes could result in the alteration of pathogenicity or antibiotic susceptibility of individual H. pylori strains. Development of a simple, rapid, and reliable screening method would be useful in the molecular characterization of genetic variation among different H. pylori strains. The copP gene from H. pylori UA802 was used as a model for developing a mutation screening method. Four point mutations were introduced into the copP gene by in vitro site-directed mutagenesis and were verified by DNA sequencing. The mutated copP gene replaced the wild-type locus by natural transformation and homologous recombination. The site-specific mutants were screened by polymerase chain reaction (PCR) using 3'-end mismatched primers. The origins of the PCR fragments were demonstrated by Southern hybridization with the copP-derived DNA probe. Three of these four mutations were characterized by PCR with the specific primers that contained the 3'-terminal nucleotide complementary only to the mutated nucleotide on both plasmid and chromosomal DNA templates. One mutation was able to be identified with the foregoing primer containing an additional wild-type nucleotide at its 3'-end. Point mutant screening with these specific primers offers 100% sensitivity in the aforementioned conditions. To achieve optimal screening, the concentration of magnesium and the annealing temperature have to be adjusted. The procedure reported in this study is a simple, economical, rapid, and efficient approach in the identification of site-specific mutations on both plasmids and chromosomal DNA. Although the method was developed by using a specified H. pylori gene, it can be extended easily to other genes of interest in H. pylori or other organisms.
-
Kim, Sobin; Ulz, Michael E; Nguyen, Tuan; Li, Chi-Ming; Sato, Takaaki; Tycko, Benjamin; Ju, Jingyue
2004-05-01
A mass spectrometry (MS) based multiplex genotyping method using solid phase capturable (SPC) dideoxynucleotides and single base extension (SBE), named the SPC-SBE, has been developed for mutation detection. We report here the simultaneous genotyping of 30 potential point mutation sites in exons 5, 7, and 8 of the human p53 gene in one tube using the SPC-SBE method. The 30 mutation sites, including the most frequently mutated p53 codons, were chosen to explore the high multiplexing scope of the SPC-SBE method. Thirty primers specific to each potential mutation site were designed to yield SBE products with sufficient mass differences. This was achieved by tuning the mass of some primers using modified nucleotides. Genomic DNA was amplified by multiplex PCR to produce amplicons of the three p53 exons. The 30 primers were combined with the PCR products and biotinylated dideoxynucleotides for SBE to generate 3'-biotinylated extension DNA products. These products were then captured by streptavidin-coated magnetic beads, while the unextended primers and other components in the reaction were washed away. The pure extension DNA products were subsequently released from the solid phase and analyzed with MS. We simultaneously genotyped 30 potential mutation sites in the p53 gene from Wilms' tumor, head and neck tumor, and colorectal tumor. Both homozygous and heterozygous genotypes were accurately determined with digital resolution. This is the highest level of multiplex genotyping reported thus far using MS, indicating that the approach might be applicable to screening a repertoire of genotypes in candidate genes as potential disease markers.
-
Recognition of the pro-mutagenic base uracil by family B DNA polymerases from archaea.
Shuttleworth, Gillian; Fogg, Mark J; Kurpiewski, Michael R; Jen-Jacobson, Linda; Connolly, Bernard A
2004-03-26
Archaeal family B DNA polymerases contain a specialised pocket that binds tightly to template-strand uracil, causing the stalling of DNA replication. The mechanism of this unique "template-strand proof-reading" has been studied using equilibrium binding measurements, DNA footprinting, van't Hoff analysis and calorimetry. Binding assays have shown that the polymerase preferentially binds to uracil in single as opposed to double-stranded DNA. Tightest binding is observed using primer-templates that contain uracil four bases in front of the primer-template junction, corresponding to the observed stalling position. Ethylation interference analysis of primer-templates shows that the two phosphates, immediately flanking the uracil (NpUpN), are important for binding; contacts are also made to phosphates in the primer-strand. Microcalorimetry and van't Hoff analysis have given a fuller understanding of the thermodynamic parameters involved in uracil recognition. All the results are consistent with a "read-ahead" mechanism, in which the replicating polymerase scans the template, ahead of the replication fork, for the presence of uracil and halts polymerisation on detecting this base. Post-stalling events, serving to eliminate uracil, await full elucidation.
-
DOE Office of Scientific and Technical Information (OSTI.GOV)
Kuppuswamy, M.N.; Hoffmann, J.W.; Spitzer, S.G.
1991-02-15
In this report, the authors describe an approach to detect the presence of abnormal alleles in those genetic diseases in which frequency of occurrence of the same mutation is high (e.g., hemophilia B). Initially, from each subject, the DNA fragment containing the putative mutation site is amplified by the polymerase chain reaction. For each fragment two reaction mixtures are then prepared. Each contains the amplified fragment, a primer (18-mer or longer) whose sequence is identical to the coding sequence of the normal gene immediately flanking the 5{prime} end of the mutation site, and either an {alpha}-{sup 32}P-labeled nucleotide corresponding tomore » the normal coding sequence at the mutation site or an {alpha}-{sup 32}P-labeled nucleotide corresponding to the mutant sequence. An essential feature of the present methodology is that the base immediately 3{prime} to the template-bound primer is one of those altered in the mutant, since in this way an extension of the primer by a single base will give an extended molecule characteristic of either the mutant or the wild type. The method is rapid and should be useful in carrier detection and prenatal diagnosis of every genetic disease with a known sequence variation.« less
-
In vitro selection using a dual RNA library that allows primerless selection
Jarosch, Florian; Buchner, Klaus; Klussmann, Sven
2006-01-01
High affinity target-binding aptamers are identified from random oligonucleotide libraries by an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX). Since the SELEX process includes a PCR amplification step the randomized region of the oligonucleotide libraries need to be flanked by two fixed primer binding sequences. These primer binding sites are often difficult to truncate because they may be necessary to maintain the structure of the aptamer or may even be part of the target binding motif. We designed a novel type of RNA library that carries fixed sequences which constrain the oligonucleotides into a partly double-stranded structure, thereby minimizing the risk that the primer binding sequences become part of the target-binding motif. Moreover, the specific design of the library including the use of tandem RNA Polymerase promoters allows the selection of oligonucleotides without any primer binding sequences. The library was used to select aptamers to the mirror-image peptide of ghrelin. Ghrelin is a potent stimulator of growth-hormone release and food intake. After selection, the identified aptamer sequences were directly synthesized in their mirror-image configuration. The final 44 nt-Spiegelmer, named NOX-B11-3, blocks ghrelin action in a cell culture assay displaying an IC50 of 4.5 nM at 37°C. PMID:16855281
-
A mutant screening method by critical annealing temperature-PCR for site-directed mutagenesis.
Liu, Ying; Wu, Ting; Song, Jian; Chen, Xuelian; Zhang, Yu; Wan, Yu
2013-03-11
Distinguishing desired mutants from parental templates and undesired mutants is a problem not well solved in Quikchange™ mutagenesis. Although Dpn I digestion can eliminate methylated parental (WT) DNA, the efficiency is not satisfying due to the existence of hemi-methylated DNA in the PCR products, which is resistant to Dpn I. The present study designed a novel critical annealing temperature (T(c))-PCR to replace Dpn I digestion for more perfect mutant distinguishing, in which part-overlapping primers containing mutation(s) were used to reduce initial concentration of template DNA in mutagenic PCR. A T(c)-PCR with the same mutagenic primers was performed without Dpn I digestion. The T(c) for each pair of the primers was identified by gradient PCR. The relationship between PCR-identified T(c) and T(m) of the primers was analyzed and modeled with correlation and regression. Gradient PCR identified a T(c) for each of 14 tested mutagenic primers, which could discriminate mismatched parental molecules and undesired mutants from desired mutants. The PCR-identified T(c) was correlated to the primer's T(m) (r = 0.804, P<0.0001). Thus, in practical applications, the T(c) can be easily calculated with a regression equation, T(c)= 48.81 + 0.253*T(m). The new protocol introduced a novel T(c)-PCR method for mutant screening which can more efficiently and accurately select against parental molecules and undesired mutations in mutagenic sequence segments.
-
Cruchaga, Carlos; Anso, Elena; Font, María; Martino, Virginia S.; Rouzaut, Ana; Martinez-Irujo, Juan J.
2007-01-01
Inhibitors of the excision reaction catalysed by HIV-1 RT (reverse transcriptase) represent a promising approach in the fight against HIV, because these molecules would interfere with the main mechanism of resistance of this enzyme towards chain-terminating nucleotides. Only a limited number of compounds have been demonstrated to inhibit this reaction to date, including NNRTIs (non-nucleoside RT inhibitors) and certain pyrophosphate analogues. We have found previously that 2GP (2-O-galloylpunicalin), an antiviral compound extracted from the leaves of Terminalia triflora, was able to inhibit both the RT and the RNase H activities of HIV-1 RT without affecting cell proliferation or viability. In the present study, we show that 2GP also inhibited the ATP- and PPi-dependent phosphorolysis catalysed by wild-type and AZT (3′-azido-3′-deoxythymidine)-resistant enzymes at sub-micromolar concentrations. Kinetic and direct-binding analysis showed that 2GP was a non-competitive inhibitor against the nucleotide substrate, whereas it competed with the binding of RT to the template–primer (Kd=85 nM). As expected from its mechanism of action, 2GP was active against mutations conferring resistance to NNRTIs and AZT. The combination of AZT with 2GP was highly synergistic when tested in the presence of pyrophosphate, indicating that the inhibition of RT-catalysed phosphorolysis was responsible for the synergy found. Although other RT inhibitors that compete with the template–primer have been described, this is the first demonstration that these compounds can be used to block the excision of chain terminating nucleotides, providing a rationale for their combination with nucleoside analogues. PMID:17355225
-
Cruchaga, Carlos; Anso, Elena; Font, María; Martino, Virginia S; Rouzaut, Ana; Martinez-Irujo, Juan J
2007-07-01
Inhibitors of the excision reaction catalysed by HIV-1 RT (reverse transcriptase) represent a promising approach in the fight against HIV, because these molecules would interfere with the main mechanism of resistance of this enzyme towards chain-terminating nucleotides. Only a limited number of compounds have been demonstrated to inhibit this reaction to date, including NNRTIs (non-nucleoside RT inhibitors) and certain pyrophosphate analogues. We have found previously that 2GP (2-O-galloylpunicalin), an antiviral compound extracted from the leaves of Terminalia triflora, was able to inhibit both the RT and the RNase H activities of HIV-1 RT without affecting cell proliferation or viability. In the present study, we show that 2GP also inhibited the ATP- and PP(i)-dependent phosphorolysis catalysed by wild-type and AZT (3'-azido-3'-deoxythymidine)-resistant enzymes at sub-micromolar concentrations. Kinetic and direct-binding analysis showed that 2GP was a non-competitive inhibitor against the nucleotide substrate, whereas it competed with the binding of RT to the template-primer (K(d)=85 nM). As expected from its mechanism of action, 2GP was active against mutations conferring resistance to NNRTIs and AZT. The combination of AZT with 2GP was highly synergistic when tested in the presence of pyrophosphate, indicating that the inhibition of RT-catalysed phosphorolysis was responsible for the synergy found. Although other RT inhibitors that compete with the template-primer have been described, this is the first demonstration that these compounds can be used to block the excision of chain terminating nucleotides, providing a rationale for their combination with nucleoside analogues.
-
DOE Office of Scientific and Technical Information (OSTI.GOV)
Ichida, Yu, E-mail: ichida-y@ncchd.go.jp; Utsunomiya, Yuko; Onodera, Masafumi
2016-03-18
Zinc finger protein 809 (ZFP809) belongs to the Kruppel-associated box-containing zinc finger protein (KRAB-ZFP) family and functions in repressing the expression of Moloney murine leukemia virus (MoMLV). ZFP809 binds to the primer-binding site (PBS)located downstream of the MoMLV-long terminal repeat (LTR) and induces epigenetic modifications at integration sites, such as repressive histone modifications and de novo DNA methylation. KRAB-ZFPs contain consensus TGEKP linkers between C2H2 zinc fingers. The phosphorylation of threonine residues within linkers leads to the inactivation of zinc finger binding to target sequences. ZFP809 also contains consensus linkers between zinc fingers. However, the function of ZFP809 linkers remainsmore » unknown. In the present study, we constructed ZFP809 proteins containing mutated linkers and examined their ability to silence transgene expression driven by MLV, binding ability to MLV PBS, and cellular localization. The results of the present study revealed that the linkers affected the ability of ZFP809 to silence transgene expression. Furthermore, this effect could be partly attributed to changes in the localization of ZFP809 proteins containing mutated linkers. Further characterization of ZFP809 linkers is required for understanding the functions and features of KRAB-ZFP-containing linkers. - Highlights: • ZFP809 has three consensus linkers between the zinc fingers. • Linkers are required for ZFP809 to silence transgene expression driven by MLV-LTR. • Linkers affect the precise nuclear localization of ZFP809.« less
-
Photoaffinity labeling of the primer binding domain in murine leukemia virus reverse transcriptase.
Tirumalai, R S; Modak, M J
1991-07-02
We have labeled the primer binding domain of murine leukemia virus reverse transcriptase (MuLV RT) by covalently cross-linking 5' end labeled d(T)8 to MuLV RT, using ultraviolet light energy. The specificity and the functional significance of the primer cross-linking reaction were demonstrated by the fact that (i) other oligomeric primers, tRNAs, and also template-primers readily compete with radiolabeled d(T)8 for the cross-linking reaction, (ii) under similar conditions, the competing primers and template-primer also inhibit the DNA polymerase activity of MuLV RT to a similar extent, (iii) substrate deoxynucleotides have no effect, and (iv) the reaction is sensitive to high ionic strength. In order to identify the primer binding domains/sites in MuLV RT; tryptic digests prepared from the covalently cross-linked MuLV RT and [32P]d(T)8 complexes were resolved on C-18 columns by reverse-phase HPLC. Three distinct radiolabeled peptides were found to contain the majority of the bound primer. Of these, peptide I contained approximately 65% radioactivity, while the remainder was associated with peptides II and III. Amino acid composition and sequence analyses of the individual peptides revealed that peptide I spans amino acid residues 72-80 in the primary amino acid sequence of MuLV RT and is located in the polymerase domain. The primer cross-linking site appears to be at or near Pro-76. Peptides II and III span amino acid residues 602-609 and 615-622, respectively, and are located in the RNase H domain. The probable cross-linking sites in peptides II and III are suggested to be at or near Leu-604 and Leu-618, respectively.
-
Aihara, Masamune; Yamamoto, Shigeru; Nishioka, Hiroko; Inoue, Yutaro; Hamano, Kimikazu; Oka, Masaaki; Mizukami, Yoichi
2012-06-15
G protein-coupled receptor 30/G protein estrogen receptor-1 (GPR30/GPER-1) is a novel membrane receptor for estrogen whose mRNA is expressed at high levels in estrogen-dependent cells such as breast cancer cell lines. However, mutations in GRP30 related to diseases remain unreported. To detect unknown mutations in the GPR30 open reading frame (ORF) quickly, the experimental conditions for high-resolution melting (HRM) analysis were examined for PCR primers, Taq polymerases, saturation DNA binding dyes, Mg(2+) concentration, and normalized temperatures. Nine known SNPs and 13 artificial point mutations within the GPR30 ORF, as well as single nucleotide variants in DNA extracted from subjects with breast cancers were tested under the optimal experimental conditions. The combination of Expand High Fidelity(PLUS) and SYTO9 in the presence of 2.0 mM MgCl(2) produced the best separation in melting curves of mutations in all regions of the GPR30 ORF. Under these experimental conditions, the mutations were clearly detected in both heterozygotes and homozygotes. HRM analysis of GPR30 using genomic DNA from subjects with breast cancers showed a novel single nucleotide variant, 111C>T in GPR30 and 4 known SNPs. The experimental conditions determined in this study for HRM analysis are useful for high throughput assays to detect unknown mutations within the GPR30 ORF. Copyright © 2012 Elsevier B.V. All rights reserved.
-
RNA-templated single-base mutation detection based on T4 DNA ligase and reverse molecular beacon.
Tang, Hongxing; Yang, Xiaohai; Wang, Kemin; Tan, Weihong; Li, Huimin; He, Lifang; Liu, Bin
2008-06-15
A novel RNA-templated single-base mutation detection method based on T4 DNA ligase and reverse molecular beacon (rMB) has been developed and successfully applied to identification of single-base mutation in codon 273 of the p53 gene. The discrimination was carried out using allele-specific primers, which flanked the variable position in the target RNA and was ligated using T4 DNA ligase only when the primers perfectly matched the RNA template. The allele-specific primers also carried complementary stem structures with end-labels (fluorophore TAMRA, quencher DABCYL), which formed a molecular beacon after RNase H digestion. One-base mismatch can be discriminated by analyzing the change of fluorescence intensity before and after RNase H digestion. This method has several advantages for practical applications, such as direct discrimination of single-base mismatch of the RNA extracted from cell; no requirement of PCR amplification; performance of homogeneous detection; and easily design of detection probes.
-
Weisshart, Klaus; Chow, Connie S.; Coen, Donald M.
1999-01-01
Herpes simplex virus DNA polymerase consists of a catalytic subunit, Pol, and a processivity subunit, UL42, that, unlike other established processivity factors, binds DNA directly. We used gel retardation and filter-binding assays to investigate how UL42 affects the polymerase-DNA interaction. The Pol/UL42 heterodimer bound more tightly to DNA in a primer-template configuration than to single-stranded DNA (ssDNA), while Pol alone bound more tightly to ssDNA than to DNA in a primer-template configuration. The affinity of Pol/UL42 for ssDNA was reduced severalfold relative to that of Pol, while the affinity of Pol/UL42 for primer-template DNA was increased ∼15-fold relative to that of Pol. The affinity of Pol/UL42 for circular double-stranded DNA (dsDNA) was reduced drastically relative to that of UL42, but the affinity of Pol/UL42 for short primer-templates was increased modestly relative to that of UL42. Pol/UL42 associated with primer-template DNA ∼2-fold faster than did Pol and dissociated ∼10-fold more slowly, resulting in a half-life of 2 h and a subnanomolar Kd. Despite such stable binding, rapid-quench analysis revealed that the rates of elongation of Pol/UL42 and Pol were essentially the same, ∼30 nucleotides/s. Taken together, these studies indicate that (i) Pol/UL42 is more likely than its subunits to associate with DNA in a primer-template configuration rather than nonspecifically to either ssDNA or dsDNA, and (ii) UL42 reduces the rate of dissociation from primer-template DNA but not the rate of elongation. Two models of polymerase-DNA interactions during replication that may explain these findings are presented. PMID:9847307
-
UK and Irish Y-STR population data-A catalogue of variant alleles.
Aliferi, Anastasia; Thomson, Jim; McDonald, Andrew; Paynter, Vanessa Molin; Ferguson, Steven; Vanhinsbergh, Des; Syndercombe Court, Denise; Ballard, David
2018-05-01
A total of 3128 Y-STR profiles from three UK and one Irish population have been analysed with the PowerPlex Y23 system and are reported here. Instances of haplotype sharing between apparently unrelated individuals were identified and further investigated with the use of the 5 additional markers within the Yfiler Plus kit, resulting in a reduction by 76% in the number of shared haplotypes. Furthermore, Yfiler Plus was also employed to verify locus deletions and duplications observed in Y23 genotypes while inconsistencies between the two kits were sequenced, revealing underlying Y23 primer binding site mutations in loci DYS392 and DYS576. Finally, the mechanism behind a previously reported population specific peak shift observed in DYS481 in South Asian samples has been evaluated and further investigated in a novel case of this phenomenon seen in a Black British individual featuring a different flanking region mutation. Copyright © 2018 Elsevier B.V. All rights reserved.
-
Primer synthesis by a eukaryotic-like archaeal primase is independent of its Fe-S cluster.
Holzer, Sandro; Yan, Jiangyu; Kilkenny, Mairi L; Bell, Stephen D; Pellegrini, Luca
2017-11-23
DNA replication depends on primase, the specialised polymerase responsible for synthesis of the RNA primers that are elongated by the replicative DNA polymerases. In eukaryotic and archaeal replication, primase is a heterodimer of two subunits, PriS and PriL. Recently, a third primase subunit named PriX was identified in the archaeon Sulfolobus solfataricus. PriX is essential for primer synthesis and is structurally related to the Fe-S cluster domain of eukaryotic PriL. Here we show that PriX contains a nucleotide-binding site required for primer synthesis, and demonstrate equivalence of nucleotide-binding residues in PriX with eukaryotic PriL residues that are known to be important for primer synthesis. A primase chimera, where PriX is fused to a truncated version of PriL lacking the Fe-S cluster domain retains wild-type levels of primer synthesis. Our evidence shows that PriX has replaced PriL as the subunit that endows primase with the unique ability to initiate nucleic acid synthesis. Importantly, our findings reveal that the Fe-S cluster is not required for primer synthesis.
-
Lu, Jennifer; Ru, Kelin; Candiloro, Ida; Dobrovic, Alexander; Korbie, Darren; Trau, Matt
2017-03-22
Multiplex bisulfite-PCR sequencing is a convenient and scalable method for the quantitative determination of the methylation state of target DNA regions. A challenge of this application is the presence of CpGs in the same region where primers are being placed. A common solution to the presence of CpGs within a primer-binding region is to substitute a base degeneracy at the cytosine position. However, the efficacy of different substitutions and the extent to which bias towards methylated or unmethylated templates may occur has never been evaluated in bisulfite multiplex sequencing applications. In response, we examined the performance of four different primer substitutions at the cytosine position of CpG's contained within the PCR primers. In this study, deoxyinosine-, 5-nitroindole-, mixed-base primers and primers with an abasic site were evaluated across a series of methylated controls. Primers that contained mixed- or deoxyinosine- base modifications performed most robustly. Mixed-base primers were further selected to determine the conditions that induce bias towards methylated templates. This identified an optimized set of conditions where the methylated state of bisulfite DNA templates can be accurately assessed using mixed-base primers, and expands the scope of bisulfite resequencing assays when working with challenging templates.
-
Silva, Felipe Carneiro; Torrezan, Giovana Tardin; Brianese, Rafael Canfield; Stabellini, Raquel; Carraro, Dirce Maria
2017-07-01
Hereditary breast and ovarian cancer is characterized by mutations in BRCA1 or BRCA2 genes and PCR-based screening techniques, such as capillary sequencing and next-generation sequencing (NGS), are considered gold standard methods for detection of pathogenic mutations in these genes. Single-nucleotide polymorphisms (SNPs) constitute a vast source of variation in the human genome and represent a risk for misdiagnosis in genetic testing, since the presence of a SNP in primer-annealing sites may cause false negative results due to allele dropout. However, few reports are available and the frequency of this phenomenon in diagnostic assays remains unknown. In this article, we investigated the causes of a false negative capillary sequencing result in BRCA1 involving a mother-daughter dyad. Using several molecular strategies, including different DNA polymerases, primer redesign, allele-specific PCR and NGS, we established that the initial misdiagnosis was caused by a SNP located in the primer-annealing region, leading to allele dropout of the mutated allele. Assuming that this problem can also occur in any PCR-based method that are widely used in diagnostic settings, the clinical report presented here draws attention for one of the limitations of genetic testing in general, for which medical and laboratory communities need to be aware.
-
Long distance PCR in detection of inversion mutations of F8C gene in hemophilia A patients.
Poláková, H; Zmetáková, I; Kádasi, L'
2003-06-01
In the present paper, the experience with detection of intron 22 inversion of F8C gene in severe hemophilia A patients using a recently described long-distance PCR (LD-PCR) method was reported. To test the sensitivity and the specifity of the LD-PCR, analysis of 46 DNA samples of patients and their family members, previously tested by Southern hybridization, was performed. In addition, 16 DNA samples of severe hemophilia A patients in which causative mutation was unknown, were included in analysis. Four-primers, P, Q, A&B, which are able to differentiate between the affected males with or without the inversion, and in female carriers, were used in LD-PCR. Two primers, P&Q, are located within the F8C gene flanking int22h1. Two further primers, A&B, flank int22h2 and int22h3, extragene homologs of int22h1. Nine combinations of four primers were used to identify the optimal one. Four-primers (P, Q, A&B), three-primers (P,Q & B;P, A & B; A, B & Q;P, Q & A) and two-primers (A & B; P & Q; A & Q; P & B) PCR amplifications were performed in the hemophilia A patients as well as in obligate carriers DNA samples. Successful amplification required introduction of some modifications of the original protocol. The most reproducible and uniform results were obtained using two-primers PCR, performed in four single reactions. Thus, a total of 46 DNA samples, 22 were hemizygous for inversion, 6 without the inversion, 14 carriers and 4 non-carriers of inversion. Perfect correlation between genotypes determined using Southern hybridization and LD-PCR was achieved. The optimalized two-primers LD-PCR protocol was used for analysis of 16 DNA samples of severe hemophilia A patients with unknown mutation. Ten cases of inversions and six cases without them were detected. Thus in additional 10 severe hemophilic patients DNA diagnosis was completed. The most successful and reproducible results were obtained performing four single LD-PCR reactions with combinations of two-primers A & B; P & Q; A&Q, and P&B in each DNA sample and this approach is recommended for routine using in clinical practice.
-
Huang, Xiaoyu; Li, Desheng; Wang, Jiwen; Huang, Yan; Han, Chunchun; Zhang, Guiquan; Huang, Zhi; Wu, Honglin; Wei, Ming; Wang, Guosong; Hu, Haiping; Deng, Tao; He, Tao; Zhou, Yingming; Song, Shixian; Luo, Bo; Zhang, Heming
2013-11-01
The different SSCP patterns of the follicle stimulating hormone beta (FSHβ) gene amplified by three pairs of primers were sequenced. Comparisons among the three nucleotide sequences of three genotypes indicated that three base substitutions (A213T, A91G, and A89C) were detected in FSHβ gene, which A213T substitution led to one amino acids mutation (Lys > Met), and the other two substitutions were synonymous mutations. The AA, AB and BB genotypes patterns obtained by FSHβ primer1 had evident relation with the litter traits, but the SSCP genotypes patterns obtained by FSHβ primer2 and primer3 had no evident relation with the litter traits in giant panda. The giant panda with AA and AB genotype had the largest litter size and multiparity rate compared with the BB genotypes (P < 0.05). We speculated that the giant pandas with the A allele have better litter traits than those with the B allele.
-
Kho, S L; Chua, K H; George, E; Tan, J A M A
2013-07-15
Beta-thalassemia is a life-threatening inherited blood disorder. Rapid characterization of β-globin gene mutations is necessary because of the high frequency of Malaysian β-thalassemia carriers. A combination real-time polymerase chain reaction genotyping assay using TaqMan probes was developed to confirm β-globin gene mutations. In this study, primers and probes were designed to specifically identify 8 common β-thalassemia mutations in the Malaysian Malay and Chinese ethnic groups using the Primer Express software. "Blind tests" using DNA samples from healthy individuals and β-thalassemia patients with different genotypes were performed to determine the specificity and sensitivity of this newly designed assay. Our results showed 100% sensitivity and specificity for this novel assay. In conclusion, the TaqMan genotyping assay is a straightforward assay that allows detection of β-globin gene mutations in less than 40 min. The simplicity and reproducibility of the TaqMan genotyping assay permit its use in laboratories as a rapid and cost-effective diagnostic tool for confirmation of common β-thalassemia mutations in Malaysia.
-
Kamau, Everlyn; Agoti, Charles N; Lewa, Clement S; Oketch, John; Owor, Betty E; Otieno, Grieven P; Bett, Anne; Cane, Patricia A; Nokes, D James
2017-03-01
Direct immuno-fluorescence test (IFAT) and multiplex real-time RT-PCR have been central to RSV diagnosis in Kilifi, Kenya. Recently, these two methods showed discrepancies with an increasing number of PCR undetectable RSV-B viruses. Establish if mismatches in the primer and probe binding sites could have reduced real-time RT-PCR sensitivity. Nucleoprotein (N) and glycoprotein (G) genes were sequenced for real-time RT-PCR positive and negative samples. Primer and probe binding regions in N gene were checked for mismatches and phylogenetic analyses done to determine molecular epidemiology of these viruses. New primers and probe were designed and tested on the previously real-time RT-PCR negative samples. N gene sequences revealed 3 different mismatches in the probe target site of PCR negative, IFAT positive viruses. The primers target sites had no mismatches. Phylogenetic analysis of N and G genes showed that real-time RT-PCR positive and negative samples fell into distinct clades. Newly designed primers-probe pair improved detection and recovered previous PCR undetectable viruses. An emerging RSV-B variant is undetectable by a quite widely used real-time RT-PCR assay due to polymorphisms that influence probe hybridization affecting PCR accuracy. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.
-
Vargas, Diana Y.; Kramer, Fred Russell; Tyagi, Sanjay; Marras, Salvatore A. E.
2016-01-01
We describe the use of “SuperSelective” primers that enable the detection and quantitation of somatic mutations whose presence relates to cancer diagnosis, prognosis, and therapy, in real-time PCR assays that can potentially analyze rare DNA fragments present in blood samples (liquid biopsies). The design of these deoxyribonucleotide primers incorporates both a relatively long “5' anchor sequence” that hybridizes strongly to target DNA fragments, and a very short, physically and functionally separate, “3' foot sequence” that is perfectly complementary to the mutant target sequence, but mismatches the wild-type sequence. As few as ten mutant fragments can reliably be detected in the presence of 1,000,000 wild-type fragments, even when the difference between the mutant and the wild type is only a single nucleotide polymorphism. Multiplex PCR assays employing a set of SuperSelective primers, and a corresponding set of differently colored molecular beacon probes, can be used in situations where the different mutations, though occurring in different cells, are located in the same codon. These non-symmetric real-time multiplex PCR assays contain limited concentrations of each SuperSelective primer, thereby enabling the simultaneous determination of each mutation’s abundance by comparing its threshold value to the threshold value of a reference gene present in the sample. PMID:27244445
-
Hill, Carolyn R; Duewer, David L; Kline, Margaret C; Sprecher, Cynthia J; McLaren, Robert S; Rabbach, Dawn R; Krenke, Benjamin E; Ensenberger, Martin G; Fulmer, Patricia M; Storts, Douglas R; Butler, John M
2011-08-01
The PowerPlex(®) ESX 17 and ESI 17 Systems for short tandem repeat (STR) amplification were developed by the Promega Corporation to meet the European Network of Forensic Science Institutes (ENFSI) and the European DNA Profiling (EDNAP) Group recommendations for increasing the number of STR loci included in the European Standard Set (ESS). The PowerPlex ESX 17 and ESI 17 Systems utilize different PCR primer combinations to co-amplify the following 17 loci: D1S1656, D2S441, D2S1338, D3S1358, D8S1179, D10S1248, D12S391, D16S539, D18S51, D19S433, D21S11, D22S1045, FGA, TH01, vWA, SE33, and the sex-typing locus amelogenin. A total of 1443 U.S. population samples were evaluated with pre-commercialization versions of both kits. Stutter and heterozygote peak height ratios have been used to characterize kit performance. Typing results have been used to estimate the match probabilities provided by the chosen loci as well as in concordance studies. Full concordance between the typing results for the two kits was observed in 99.994% (49,055 out of 49,062) STR allele calls compared. All genotyping discrepancies were confirmed by DNA sequence analysis. As a result of these comparisons, a second forward primer for the D22S1045 locus has been added to the PowerPlex ESX 17 System to address a primer binding site mutation and the D1S1656 locus reverse primer in the PowerPlex ESI 17 System was modified to eliminate an amplification-efficiency reducing primer dimer. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.
-
Genome-Wide Epigenetic Studies in Human Disease: A Primer on -Omic Technologies
Yan, Huihuang; Tian, Shulan; Slager, Susan L.; Sun, Zhifu; Ordog, Tamas
2016-01-01
Epigenetic information encoded in covalent modifications of DNA and histone proteins regulates fundamental biological processes through the action of chromatin regulators, transcription factors, and noncoding RNA species. Epigenetic plasticity enables an organism to respond to developmental and environmental signals without genetic changes. However, aberrant epigenetic control plays a key role in pathogenesis of disease. Normal epigenetic states could be disrupted by detrimental mutations and expression alteration of chromatin regulators or by environmental factors. In this primer, we briefly review the epigenetic basis of human disease and discuss how recent discoveries in this field could be translated into clinical diagnosis, prevention, and treatment. We introduce platforms for mapping genome-wide chromatin accessibility, nucleosome occupancy, DNA-binding proteins, and DNA methylation, primarily focusing on the integration of DNA methylation and chromatin immunoprecipitation–sequencing technologies into disease association studies. We highlight practical considerations in applying high-throughput epigenetic assays and formulating analytical strategies. Finally, we summarize current challenges in sample acquisition, experimental procedures, data analysis, and interpretation and make recommendations on further refinement in these areas. Incorporating epigenomic testing into the clinical research arsenal will greatly facilitate our understanding of the epigenetic basis of disease and help identify novel therapeutic targets. PMID:26721890
-
Pena, S D; Barreto, G; Vago, A R; De Marco, L; Reinach, F C; Dias Neto, E; Simpson, A J
1994-01-01
Low-stringency single specific primer PCR (LSSP-PCR) is an extremely simple PCR-based technique that detects single or multiple mutations in gene-sized DNA fragments. A purified DNA fragment is subjected to PCR using high concentrations of a single specific oligonucleotide primer, large amounts of Taq polymerase, and a very low annealing temperature. Under these conditions the primer hybridizes specifically to its complementary region and nonspecifically to multiple sites within the fragment, in a sequence-dependent manner, producing a heterogeneous set of reaction products resolvable by electrophoresis. The complex banding pattern obtained is significantly altered by even a single-base change and thus constitutes a unique "gene signature." Therefore LSSP-PCR will have almost unlimited application in all fields of genetics and molecular medicine where rapid and sensitive detection of mutations and sequence variations is important. The usefulness of LSSP-PCR is illustrated by applications in the study of mutants of smooth muscle myosin light chain, analysis of a family with X-linked nephrogenic diabetes insipidus, and identity testing using human mitochondrial DNA. Images PMID:8127912
-
[Construction of thr461 --> Asn461 and Ile462 --> Val462 mutation vector of P4501A1 gene].
Wei, Qing; Liu, Yi-Min; Wang, Hui; Zhao, Xiao-Lin; Ren, Tie-ling; Xiao, Yong-mei
2006-09-01
To construct Thr461 --> Asn461 and Ile462 --> Val462 mutation vector of P4501A1 gene and to provide scientific base for deeply researching on the function of cytochrome 1A1 gene (CYP1A1) and the mechanism of carcinogenesis. According to cDNA sequence of human CYP1A1 gene, universal primers (Pm3/Pm4) and mutant primers (Pt15/Pt16 and Pt17/Pt18) containing restriction enzyme site and mutation site were designed. The first set of primers involving Pm3/Pt16 and Pm3/Pt18 amplified a forward 1.5kb fragment from pGEM-T-CYP1A1 plasmid. The second set of primers involving Pt15/Pm4 and Pt17/Pm4 amplified a reverse 177-bp fragment from 10ng pGEM-T-CYP1A1 plasmid. The third set of primers involving Pm3/Pm4 amplified a 1.5kb fragment from the fomer PCR amplifications. The third PCR products were separated, purified and recovered from 1% agarose gel, then inserted into pMD-T vector. Subsequently the conjunct products were transformed into E. coil strain DH-5alpha., then the single clone was screened out and plasmids were extracted from such clone finally verified by restriction endonuclease analysis and sequencing. A 1.5kb fragment of tricycle PCR amplifications were digested by restriction endonucleases (BamHI and SailI) and sequenced bidirectionally by universal primers(T7p and SP6). The results verified that the cloned fragment including Asn461 and Val462 mutant site had 99.9% homology with the human cDNA of CYP1A1 gene in Genebank. The objective fragment containing Asn461 and Va462 mutant site with cDNA of the CYP1A1 gene has been successfully constructed in this experiment.
-
Reddy, G; Nanduri, V B; Basu, A; Modak, M J
1991-08-20
Treatment of murine leukemia virus reverse transcriptase (MuLV RT) with potassium ferrate, an oxidizing agent known to oxidize amino acids involved in phosphate binding domains of proteins, results in the irreversible inactivation of both the DNA polymerase and the RNase H activities. Significant protection from ferrate-mediated inactivation is observed in the presence of template-primer but not in the presence of substrate deoxynucleoside triphosphates. Furthermore, ferrate-treated enzyme loses template-primer binding activity as judged by UV-mediated cross-linking of radiolabeled DNA. Comparative tryptic peptide mapping by reverse-phase HPLC of native and ferrate-oxidized enzyme indicated the presence of two new peptides eluting at 38 and 57 min and a significant loss of a peptide eluting at 74 min. Purification, amino acid composition, and sequencing of these affected peptides revealed that they correspond to amino acid residues 285-295, 630-640, and 586-599, respectively, in the primary amino acid sequence of MuLV RT. These results indicate that the domains constituted by the above peptides are important for the template-primer binding function in MuLV RT. Peptide I is located in the polymerase domain whereas peptides II and III are located in the RNase H domain. Amino acid sequence analysis of peptides I and II suggested Lys-285 and Cys-635 as the probable sites of ferrate action.
-
Anwar, R; Booth, A; Churchill, A J; Markham, A F
1996-01-01
The determination of nucleotide sequence is fundamental to the identification and molecular analysis of genes. Direct sequencing of PCR products is now becoming a commonplace procedure for haplotype analysis, and for defining mutations and polymorphism within genes, particularly for diagnostic purposes. A previously unrecognised phenomenon, primer related variability, observed in sequence data generated using Taq cycle sequencing and T7 Sequenase sequencing, is reported. This suggests that caution is necessary when interpreting DNA sequence data. This is particularly important in situations where treatment may be dependent on the accuracy of the molecular diagnosis. Images PMID:16696096
-
Reverse transcription of phage RNA and its fragment directed by synthetic heteropolymeric primers
Frolova, L. Yu.; Metelyev, V. G.; Ratmanova, K. I.; Smirnov, V. D.; Shabarova, Z. A.; Prokofyev, M. A.; Berzin, V. M.; Jansone, I. V.; Gren, E. J.; Kisselev, L. L.
1977-01-01
DNA synthesis catalysed by RNA-directed DNA-polymerase (reverse transcriptase) was found to proceed on the RNA template of an MS2 phage in the presence of heteropolymeric synthetic octa- and nonadeoxyribonucleotide primers complementary to the intercistronic region (coat protein binding site) and the region of the coat protein cistron, respectively. The product of synthesis consists of discrete DNA fractions of different length, including transcripts longer than 1,000 nucleotides. The coat protein inhibits DNA synthesis if it is initiated at its binding site, but has no effect on DNA synthesis initiated at the coat protein cistron. It has been suggested that, in this system, the initiation of DNA synthesis by synthetic primers is topographically specific. The MS2 coat protein binding site (an RNA fragment of 59 nucleotides) serves as a template for polydeoxyribonucleotide synthesis in the presence of octanucleotide primer and reverse transcriptase. The product of synthesis is homogenous and its length corresponds to the length of the template. The effective and complete copying of the fragment having a distinct secondary structure proves that the secondary structure does not interfere, in principle, with RNA being a template in the system of reverse transcription. PMID:71713
-
Ryden, T A; de Mars, M; Beemon, K
1993-01-01
Several C/EBP binding sites within the Rous sarcoma virus (RSV) long terminal repeat (LTR) and gag enhancers were mutated, and the effect of these mutations on viral gene expression was assessed. Minimal site-specific mutations in each of three adjacent C/EBP binding sites in the LTR reduced steady-state viral RNA levels. Double mutation of the two 5' proximal LTR binding sites resulted in production of 30% of wild-type levels of virus. DNase I footprinting analysis of mutant DNAs indicated that the mutations blocked C/EBP binding at the affected sites. Additional C/EBP binding sites were identified upstream of the 3' LTR and within the 5' end of the LTRs. Point mutations in the RSV gag intragenic enhancer region, which blocked binding of C/EBP at two of three adjacent C/EBP sites, also reduced virus production significantly. Nuclear extracts prepared from both chicken embryo fibroblasts (CEFs) and chicken muscle contained proteins binding to the same RSV DNA sites as did C/EBP, and mutations that prevented C/EBP binding also blocked binding of these chicken proteins. It appears that CEFs and chicken muscle contain distinct proteins binding to these RSV DNA sites; the CEF binding protein was heat stable, as is C/EBP, while the chicken muscle protein was heat sensitive. Images PMID:8386280
-
Alcaide, Miguel; Liu, Mark
2013-01-01
Genes of the Major Histocompatibility Complex (MHC) have become an important marker for the investigation of adaptive genetic variation in vertebrates because of their critical role in pathogen resistance. However, despite significant advances in the last few years the characterization of MHC variation in non-model species still remains a challenging task due to the redundancy and high variation of this gene complex. Here we report the utility of a single pair of primers for the cross-amplification of the third exon of MHC class I genes, which encodes the more polymorphic half of the peptide-binding region (PBR), in oscine passerines (songbirds; Aves: Passeriformes), a group especially challenging for MHC characterization due to the presence of large and complex MHC multigene families. In our survey, although the primers failed to amplify exon 3 from two suboscine passerine birds, they amplified exon 3 of multiple MHC class I genes in all 16 species of oscine songbirds tested, yielding a total of 120 sequences. The 16 songbird species belong to 14 different families, primarily within the Passerida, but also in the Corvida. Using a conservative approach based on the analysis of cloned amplicons (n = 16) from each species, we found between 3 and 10 MHC sequences per individual. Each allele repertoire was highly divergent, with the overall number of polymorphic sites per species ranging from 33 to 108 (out of 264 sites) and the average number of nucleotide differences between alleles ranging from 14.67 to 43.67. Our survey in songbirds allowed us to compare macroevolutionary dynamics of exon 3 between songbirds and non-passerine birds. We found compelling evidence of positive selection acting specifically upon peptide-binding codons across birds, and we estimate the strength of diversifying selection in songbirds to be about twice that in non-passerines. Analysis using comparative methods suggest weaker evidence for a higher GC content in the 3rd codon position of exon 3 in non-passerine birds, a pattern that contrasts with among-clade GC patterns found in other avian studies and may suggests different mutational mechanisms. Our primers represent a useful tool for the characterization of functional and evolutionarily relevant MHC variation across the hyperdiverse songbirds. PMID:23781408
-
DOE Office of Scientific and Technical Information (OSTI.GOV)
Brown, Alistair K.; Sridharan, Sudharsan; Kremer, Laurent
Mycolic acids are the dominant feature of the Mycobacterium tuberculosis cell wall. These {alpha}-alkyl, {beta}-hydroxy fatty acids are formed by the condensation of two fatty acids, a long meromycolic acid and a shorter C{sub 24}-C{sub 26} fatty acid. The component fatty acids are produced via a combination of type I and II fatty acid synthases (FAS) with FAS-I products being elongated by FAS-II toward meromycolic acids. The {beta}-ketoacyl-acyl carrier protein (ACP) synthase III encoded by mtfabH (mtFabH) links FAS-I and FAS-II, catalyzing the condensation of FAS-I-derived acyl-CoAs with malonyl-acyl carrier protein (ACP). The acyl-CoA chain length specificity of mtFabH wasmore » assessed in vitro; the enzyme extended longer, physiologically relevant acyl-CoA primers when paired with AcpM, its natural partner, than with Escherichia coli ACP. The ability of the enzyme to use E. coli ACP suggests that a similar mode of binding is likely with both ACPs, yet it is clear that unique factors inherent to AcpM modulate the substrate specificity of mtFabH. Mutation of proposed key mtFabH residues was used to define their catalytic roles. Substitution of supposed acyl-CoA binding residues reduced transacylation, with double substitutions totally abrogating activity. Mutation of Arg{sup 46} revealed its more critical role in malonyl-AcpM decarboxylation than in the acyl-CoA binding role. Interestingly, this effect was suppressed intragenically by Arg{sup 161} {yields} Ala substitution. Our structural studies suggested that His{sup 258}, previously implicated in malonyl-ACP decarboxylation, also acts as an anchor point for a network of water molecules that we propose promotes deprotonation and transacylation of Cys{sup 122}.« less
-
Mehndiratta, Mohit; Palanichamy, Jayanth Kumar; Ramalingam, Pradeep; Pal, Arnab; Das, Prerna; Sinha, Subrata; Chattopadhyay, Parthaprasad
2008-12-01
Quantitative real-time PCR (qPCR) is a standard method used for quantification of specific gene expression. This utilizes either dsDNA binding dyes or probe based chemistry. While dsDNA binding dyes have the advantage of low cost and flexibility, fluorescence due to primer dimers also interferes with the fluorescence of the specific product. Sometimes it is difficult, if not impossible, to standardize conditions and redesign primers in such a way that only specific fluorescence of the products of test and reference genes are acquired. Normally, the fluorescence acquisition in qPCR using dsDNA binding dyes is done during the melting phase of the PCR at a temperature between the melting points of primer dimers and the specific product. We have modified the protocol to acquire fluorescence during the hybridization phase. This significantly increased the signal-to-noise ratio and enabled the use of dsDNA binding dyes for mRNA quantification in situations where it was not possible when measurement was done in the melting phase. We have demonstrated it for three mRNAs, E6, E7, and DNMT1 with beta-actin as the reference gene, and for two miRNAs. This modification broadens the scope of qPCR using dsDNA binding dyes.
-
Influenza A virus hemagglutinin glycosylation compensates for antibody escape fitness costs.
Kosik, Ivan; Ince, William L; Gentles, Lauren E; Oler, Andrew J; Kosikova, Martina; Angel, Matthew; Magadán, Javier G; Xie, Hang; Brooke, Christopher B; Yewdell, Jonathan W
2018-01-01
Rapid antigenic evolution enables the persistence of seasonal influenza A and B viruses in human populations despite widespread herd immunity. Understanding viral mechanisms that enable antigenic evolution is critical for designing durable vaccines and therapeutics. Here, we utilize the primerID method of error-correcting viral population sequencing to reveal an unexpected role for hemagglutinin (HA) glycosylation in compensating for fitness defects resulting from escape from anti-HA neutralizing antibodies. Antibody-free propagation following antigenic escape rapidly selected viruses with mutations that modulated receptor binding avidity through the addition of N-linked glycans to the HA globular domain. These findings expand our understanding of the viral mechanisms that maintain fitness during antigenic evolution to include glycan addition, and highlight the immense power of high-definition virus population sequencing to reveal novel viral adaptive mechanisms.
-
Yu, Tao; Wang, Xiao-Qing; Sang, Jian-Ping; Pan, Chun-Xu; Zou, Xian-Wu; Chen, Tsung-Yu; Zou, Xiaoqin
2012-01-01
Mutations in ClC channel proteins may cause serious functional changes and even diseases. The function of ClC proteins mainly manifests as Cl− transport, which is related to the binding free energies of chloride ions. Therefore, the influence of a mutation on ClC function can be studied by investigating the mutational effect on the binding free energies of chloride ions. The present study provides quantitative and systematic investigations on the influences of residue mutations on the electrostatic binding free energies in Escherichia coli ClC (EcClC) proteins, using all-atom molecular dynamics simulations. It was found that the change of the electrostatic binding free energy decreases linearly with the increase of the residue-chloride ion distance for a mutation. This work reveals how changes in the charge of a mutated residue and in the distance between the mutated residue and the binding site govern the variations in the electrostatic binding free energies, and therefore influence the transport of chloride ions and conduction in EcClC. This work would facilitate our understanding of the mutational effects on transport of chloride ions and functions of ClC proteins, and provide a guideline to estimate which residue mutations will have great influences on ClC functions. PMID:22612693
-
Space environment induced mutations prefer to occur at polymorphic sites of rice genomes
NASA Astrophysics Data System (ADS)
Li, Y.; Liu, M.; Cheng, Z.; Sun, Y.
To explore the genomic characteristics of rice mutants induced by space environment, space-induced mutants 971-5, 972-4, and R955, which acquired new traits after space flight such as increased yield, reduced resistance to rice blast, and semi-dwarfism compared with their on-ground controls, 971ck, 972ck, and Bing95-503, respectively, together with other 8 japonica and 3 indica rice varieties, 17 in total, were analyzed by amplified fragment length polymorphism (AFLP) method. We chose 16 AFLP primer-pairs which generated a total of 1251 sites, of which 745 (59.6%) were polymorphic over all the genotypes. With the 16 pairs of primer combinations, 54 space-induced mutation sites were observed in 971-5, 86 in 972-4, and 5 in R955 compared to their controls, and the mutation rates were 4.3%, 6.9% and 0.4%, respectively. Interestingly, 75.9%, 84.9% and 100% of the mutation sites identified in 971-5, 972-4, and R955 occurred in polymorphic sites. This result suggests that the space environment preferentially induced mutations at polymorphic sites in rice genomes and might share a common mechanism with other types of mutagens. It also implies that polymorphic sites in genomes are potential "hotspots" for mutations induced by the space environment.
-
Genotyping of K-ras codons 12 and 13 mutations in colorectal cancer by capillary electrophoresis.
Chen, Yen-Ling; Chang, Ya-Sian; Chang, Jan-Gowth; Wu, Shou-Mei
2009-06-26
Point mutations of the K-ras gene located in codons 12 and 13 cause poor responses to the anti-epidermal growth factor receptor (anti-EGFR) therapy of colorectal cancer (CRC) patients. Besides, mutations of K-ras gene have also been proven to play an important role in human tumor progression. We established a simple and effective capillary electrophoresis (CE) method for simultaneous point mutation detection in codons 12 and 13 of K-ras gene. We combined one universal fluorescence-based nonhuman-sequence primer and two fragment-oriented primers in one tube, and performed this two-in-one polymerase chain reaction (PCR). PCR fragments included wild type and seven point mutations at codons 12 and 13 of K-ras gene. The amplicons were analyzed by single-strand conformation polymorphism (SSCP)-CE method. The CE analysis was performed by using a 1x Tris-borate-EDTA (TBE) buffer containing 1.5% (w/v) hydroxyethylcellulose (HEC) (MW 250,000) under reverse polarity with 15 degrees C and 30 degrees C. Ninety colorectal cancer patients were blindly genotyped using this developed method. The results showed good agreement with those of DNA sequencing method. The SSCP-CE was feasible for mutation screening of K-ras gene in populations.
-
Primer Extension Mutagenesis Powered by Selective Rolling Circle Amplification
Huovinen, Tuomas; Brockmann, Eeva-Christine; Akter, Sultana; Perez-Gamarra, Susan; Ylä-Pelto, Jani; Liu, Yuan; Lamminmäki, Urpo
2012-01-01
Primer extension mutagenesis is a popular tool to create libraries for in vitro evolution experiments. Here we describe a further improvement of the method described by T.A. Kunkel using uracil-containing single-stranded DNA as the template for the primer extension by additional uracil-DNA glycosylase treatment and rolling circle amplification (RCA) steps. It is shown that removal of uracil bases from the template leads to selective amplification of the nascently synthesized circular DNA strand carrying the desired mutations by phi29 DNA polymerase. Selective RCA (sRCA) of the DNA heteroduplex formed in Kunkel's mutagenesis increases the mutagenesis efficiency from 50% close to 100% and the number of transformants 300-fold without notable diversity bias. We also observed that both the mutated and the wild-type DNA were present in at least one third of the cells transformed directly with Kunkel's heteroduplex. In contrast, the cells transformed with sRCA product contained only mutated DNA. In sRCA, the complex cell-based selection for the mutant strand is replaced with the more controllable enzyme-based selection and less DNA is needed for library creation. Construction of a gene library of ten billion members is demonstrated with the described method with 240 nanograms of DNA as starting material. PMID:22355397
-
Identification of the sequence variations of 15 autosomal STR loci in a Chinese population.
Chen, Wenjing; Cheng, Jianding; Ou, Xueling; Chen, Yong; Tong, Dayue; Sun, Hongyu
2014-01-01
DNA sequence variation including base(s) changes and insertion or deletion in the primer binding region may cause a null allele and, if this changes the length of the amplified fragment out of the allelic ladder, off-ladder (OL) alleles may be detected. In order to provide accurate and reliable DNA evidence for forensic DNA analysis, it is essential to clarify sequence variations in prevalently used STR loci. Suspected null alleles and OL alleles of PlowerPlex16® System from 21,934 unrelated Chinese individuals were verified by alternative systems and sequenced. A total of 17 cases with null alleles were identified, including 12 kinds of point mutations in 16 cases and a 19-base deletion in one case. The total frequency of null alleles was 7.751 × 10(-4). Eight hundred and forty-four OL alleles classified as being of 97 different kinds were observed at 15 STR loci of the PowerPlex®16 system except vWA. All the frequencies of OL alleles were under 0.01. Null alleles should be confirmed by alternative primers and OL alleles should be named appropriately. Particular attention should be paid to sequence variation, since incorrect designation could lead to false conclusions.
-
Chang, Ken C N; Galuska, Stefan; Weiner, Russell; Marton, Matthew J
2013-01-01
Somatic mutations identified on genes related to the cancer-developing signaling pathways have drawn attention in the field of personalized medicine in recent years. Treatments developed to target a specific signaling pathway may not be effective when tumor activating mutations occur downstream of the target and bypass the targeted mechanism. For instance, mutations detected in KRAS/BRAF/NRAS genes can lead to EGFR-independent intracellular signaling pathway activation. Most patients with these mutations do not respond well to anti-EGFR treatment. In an effort to detect various mutations in FFPE tissue samples among multiple solid tumor types for patient stratification many mutation assays were evaluated. Since there were more than 30 specific mutations among three targeted RAS/RAF oncogenes that could activate MAPK pathway genes, a custom designed Single Nucleotide Primer Extension (SNPE) multiplexing mutation assay was developed and analytically validated as a clinical trial assay. Throughout the process of developing and validating the assay we overcame many technical challenges which include: the designing of PCR primers for FFPE tumor tissue samples versus normal blood samples, designing of probes for detecting consecutive nucleotide double mutations, the kinetics and thermodynamics aspects of probes competition among themselves and against target PCR templates, as well as validating an assay when positive control tumor tissue or cell lines with specific mutations are not available. We used Next Generation sequencing to resolve discordant calls between the SNPE mutation assay and Sanger sequencing. We also applied a triplicate rule to reduce potential false positives and false negatives, and proposed special considerations including pre-define a cut-off percentage for detecting very low mutant copies in the wild-type DNA background.
-
Abnormal expression and mutation of p53 in cervical cancer--a study at protein, RNA and DNA levels.
Ngan, H Y; Tsao, S W; Liu, S S; Stanley, M
1997-02-01
The objectives of this study are to document the status of p53 expression and mutation in cervical cancer at protein, RNA and DNA levels and to relate this to the presence of HPV. Biopsy specimens from one hundred and three squamous cell carcinoma of the cervix and histologically normal ectocervix were analysed. Fresh tissues were extracted for protein, RNA and DNA and flash frozen tissue cryostat sectioned for immunohistochemical staining. HPV DNA status was determined by PCR using L1 consensus primers and typed for HPV 16 and 18 with E6 specific primers. p53 expression was determined at the protein level by Western blotting on protein extracts and at RNA level by Northern blotting. There was no p53 overexpression or mutation detectable in the protein extracts. Three of 65 (4.6%) of the carcinomas were positive for p53 by immunostaining with the polyclonal antibody CM1. Overexpression at the RNA level was detected in 2 of 32 (6.3%) carcinomas. p53 mutation was screened for by PCR/SSCP (single strand conformation polymorphism) followed by sequencing to define the site of mutation. Two of the cervical cancers (2.0%) showed mutation in p53 in exons 7 or 8. The mutation rate in HPV positive tumours was 1.2% (1/81) and in HPV negative tumours was 5.2% (1/19). p53 overexpression or mutation does not seem to play a significant role in cervical carcinomas.
-
Nakayama, Manabu; Oda, Hirotsugu; Nakagawa, Kenji; Yasumi, Takahiro; Kawai, Tomoki; Izawa, Kazushi; Nishikomori, Ryuta; Heike, Toshio; Ohara, Osamu
2017-03-01
Autoinflammatory diseases occupy one of a group of primary immunodeficiency diseases that are generally thought to be caused by mutation of genes responsible for innate immunity, rather than by acquired immunity. Mutations related to autoinflammatory diseases occur in 12 genes. For example, low-level somatic mosaic NLRP3 mutations underlie chronic infantile neurologic, cutaneous, articular syndrome (CINCA), also known as neonatal-onset multisystem inflammatory disease (NOMID). In current clinical practice, clinical genetic testing plays an important role in providing patients with quick, definite diagnoses. To increase the availability of such testing, low-cost high-throughput gene-analysis systems are required, ones that not only have the sensitivity to detect even low-level somatic mosaic mutations, but also can operate simply in a clinical setting. To this end, we developed a simple method that employs two-step tailed PCR and an NGS system, MiSeq platform, to detect mutations in all coding exons of the 12 genes responsible for autoinflammatory diseases. Using this amplicon sequencing system, we amplified a total of 234 amplicons derived from the 12 genes with multiplex PCR. This was done simultaneously and in one test tube. Each sample was distinguished by an index sequence of second PCR primers following PCR amplification. With our procedure and tips for reducing PCR amplification bias, we were able to analyze 12 genes from 25 clinical samples in one MiSeq run. Moreover, with the certified primers designed by our short program-which detects and avoids common SNPs in gene-specific PCR primers-we used this system for routine genetic testing. Our optimized procedure uses a simple protocol, which can easily be followed by virtually any office medical staff. Because of the small PCR amplification bias, we can analyze simultaneously several clinical DNA samples with low cost and can obtain sufficient read numbers to detect a low level of somatic mosaic mutations.
-
Matamoros, Tania; Barrioluengo, Verónica; Abia, David; Menéndez-Arias, Luis
2013-12-23
At high temperatures, RNA denaturation can improve the efficiency and specificity of reverse transcription. Refined structures and molecular models of HIV-1 reverse transcriptases (RTs) from phylogenetically distant clades (i.e., group M subtype B and group O) revealed a major interaction between the template-primer and the Arg³⁵⁸-Gly³⁵⁹-Ala³⁶⁰ triad in the large subunit of HIV-1M/B RT. However, fewer contacts were predicted for the equivalent Lys³⁵⁸-Ala³⁵⁹-Ser³⁶⁰ triad of HIV-1O RT and the nucleic acid. An engineered HIV-1O K358R/A359G/S360A RT showed increased cDNA synthesis efficiency above 68 °C, as determined by qualitative and quantitative reverse transcription polymerase chain reactions. In comparison with wild-type HIV-1O RT, the mutant enzyme showed higher thermal stability but retained wild-type RNase H activity. Mutations that increased the accuracy of HIV-1M/B RTs were tested in combination with the K358R/A359G/S360A triple mutation. Some of them (e.g., F61A, K65R, K65R/V75I, and V148I) had a negative effect on reverse transcription efficiency above 65 °C. RTs with improved DNA binding affinities also showed higher cDNA synthesis efficiencies at elevated temperatures. Two of the most thermostable RTs (i.e., mutants T69SSG/K358R/A359G/S360A and K358R/A359G/S360A/E478Q) showed moderately increased fidelity in forward mutation assays. Our results demonstrate that the triad of Arg³⁵⁸, Gly³⁵⁹, and Ala³⁶⁰ in the major groove binding track of HIV-1 RT is a major target for RT stabilization, and most relevant for improving reverse transcription efficiency at high temperatures.
-
Pham-Ledard, Anne; Prochazkova-Carlotti, Martina; Deveza, Mélanie; Laforet, Marie-Pierre; Beylot-Barry, Marie; Vergier, Béatrice; Parrens, Marie; Feuillard, Jean; Merlio, Jean-Philippe; Gachard, Nathalie
2017-11-01
Immunophenotype of primary cutaneous diffuse large B-cell lymphoma, leg-type (PCLBCL-LT) suggests a germinal center-experienced B lymphocyte (BCL2+ MUM1+ BCL6+/-). As maturation history of B-cell is "imprinted" during B-cell development on the immunoglobulin gene sequence, we studied the structure and sequence of the variable part of the genes (IGHV, IGLV, IGKV), immunoglobulin surface expression and features of class switching in order to determine the PCLBCL-LT cell of origin. Clonality analysis with BIOMED2 protocol and VH leader primers was done on DNA extracted from frozen skin biopsies on retrospective samples from 14 patients. The clonal DNA IGHV sequence of the tumor was aligned and compared with the closest germline sequence and homology percentage was calculated. Superantigen binding sites were studied. Features of selection pressure were evaluated with the multinomial Lossos model. A functional monoclonal sequence was observed in 14 cases as determined for IGHV (10), IGLV (2) or IGKV (3). IGV mutation rates were high (>5%) in all cases but one (median:15.5%), with superantigen binding sites conservation. Features of selection pressure were identified in 11/12 interpretable cases, more frequently negative (75%) than positive (25%). Intraclonal variation was detected in 3 of 8 tumor specimens with a low rate of mutations. Surface immunoglobulin was an IgM in 12/12 cases. FISH analysis of IGHM locus, deleted during class switching, showed heterozygous IGHM gene deletion in half of cases. The genomic PCR analysis confirmed the deletions within the switch μ region. IGV sequences were highly mutated but functional, with negative features of selection pressure suggesting one or more germinal center passage(s) with somatic hypermutation, but superantigen (SpA) binding sites conservation. Genetic features of class switch were observed, but on the non functional allele and co-existing with primary isotype IgM expression. These data suggest that cell-of origin is germinal center experienced and superantigen driven selected B-cell, in a stage between germinal center B-cell and plasma cell. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.
-
Chat-Uthai, Nunthawut; Vejvisithsakul, Pichpisith; Udommethaporn, Sutthirat; Meesiri, Puttarakun; Danthanawanit, Chetiya; Wongchai, Yannawan; Teerapakpinyo, Chinachote; Shuangshoti, Shanop; Poungvarin, Naravat
2018-01-01
The protein kinase BRAF is one of the key players in regulating cellular responses to extracellular signals. Somatic mutations of the BRAF gene, causing constitutive activation of BRAF, have been found in various types of human cancers such as malignant melanoma, and colorectal cancer. BRAF V600E and V600K, most commonly observed mutations in these cancers, may predict response to targeted therapies. Many techniques suffer from a lack of diagnostic sensitivity in mutation analysis in clinical samples with a low cancer cell percentage or poor-quality fragmented DNA. Here we present allele-specific real-time PCR assay for amplifying 35- to 45-base target sequences in BRAF gene. Forward primer designed for BRAF V600E detection is capable of recognizing both types of BRAF V600E mutation, i.e. V600E1 (c.1799T>A) and V600E2 (c.1799_1800delTGinsAA), as well as complex tandem mutation caused by nucleotide changes in codons 600 and 601. We utilized this assay to analyze Thai formalin-fixed paraffin-embedded tissues. Forty-eight percent of 178 Thai colorectal cancer tissues has KRAS mutation detected by highly sensitive commercial assays. Although these DNA samples contain low overall yield of amplifiable DNA, our newly-developed assay successfully revealed BRAF V600 mutations in 6 of 93 formalin-fixed paraffin-embedded colorectal cancer tissues which KRAS mutation was not detected. Ultra-short PCR assay with forward mutation-specific primers is potentially useful to detect BRAF V600 mutations in highly fragmented DNA specimens from cancer patients.
-
Parasuraman, Ponnusamy; Murugan, Veeramani; Selvin, Jeyasigamani F A; Gromiha, M Michael; Fukui, Kazuhiko; Veluraja, Kasinadar
2014-08-01
Wheat germ agglutinin (WGA) is a plant lectin, which specifically recognizes the sugars NeuNAc and GlcNAc. Mutated WGA with enhanced binding specificity can be used as biomarkers for cancer. In silico mutations are performed at the active site of WGA to enhance the binding specificity towards sialylglycans, and molecular dynamics simulations of 20 ns are carried out for wild type and mutated WGAs (WGA1, WGA2, and WGA3) in complex with sialylgalactose to examine the change in binding specificity. MD simulations reveal the change in binding specificity of wild type and mutated WGAs towards sialylgalactose and bound conformational flexibility of sialylgalactose. The mutated polar amino acid residues Asn114 (S114N), Lys118 (G118K), and Arg118 (G118R) make direct and water mediated hydrogen bonds and hydrophobic interactions with sialylgalactose. An analysis of possible hydrogen bonds, hydrophobic interactions, total pair wise interaction energy between active site residues and sialylgalactose and MM-PBSA free energy calculation reveals the plausible binding modes and the role of water in stabilizing different binding modes. An interesting observation is that the binding specificity of mutated WGAs (cyborg lectin) towards sialylgalactose is found to be higher in double point mutation (WGA3). One of the substituted residues Arg118 plays a crucial role in sugar binding. Based on the interactions and energy calculations, it is concluded that the order of binding specificity of WGAs towards sialylgalactose is WGA3 > WGA1 > WGA2 > WGA. On comparing with the wild type, double point mutated WGA (WGA3) exhibits increased specificity towards sialylgalactose, and thus, it can be effectively used in targeted drug delivery and as biological cell marker in cancer therapeutics. Copyright © 2014 John Wiley & Sons, Ltd.
-
Winn-Deen
1998-12-01
Background: Currently analysis of point mutations can be done by allele-specific polymerase chain reaction (PCR) followed by gel analysis or by gene-specific PCR followed by hybridization with an allele-specific probe. Both of these mutation detection methods require post-PCR laboratory time and run the risk of contaminating subsequent experiments with the PCR product liberated during the detection step. The author has combined the PCR amplification and detection steps into a single procedure suitable for closed-tube analysis. Methods and Results: Allele-specific PCR primers were designed as Sunrise energy-transfer primers and contained a 3' terminal mismatch to distinguish between normal and mutant DNA. Cloned normal (W64) and mutant (R64) templates of the beta3-adrenergic receptor gene were tested to verify amplification specificity and yield. A no-target negative control was also run with each reaction. After PCR, each reaction was tested for fluorescence yield by measuring fluorescence on a spectrofluorimeter or fluorescent microtitreplate reader. The cloned controls and 24 patient samples were tested for the W64R mutation by two methods. The direct fluorescence results with the Sunrise allele-specific PCR method gave comparable genotypes to those obtained with the PCR/ restriction digest/gel electrophoresis control method. No PCR artifacts were observed in the negative controls or in the PCR reactions run with the mismatched target. Conclusions: The results of this pilot study indicate good PCR product and fluorescence yield from allele-specific energy-transfer labeled primers, and the capability of distinguishing between normal and mutant alleles based on fluorescence alone, without the need for restriction digestion, gel electrophoresis, or hybridization with an allele-specific probe.
-
In the Thick of It: HCM-Causing Mutations in Myosin Binding Proteins of the Thick Filament
Harris, Samantha P.; Lyons, Ross G.; Bezold, Kristina L.
2010-01-01
In the 20 yrs since the discovery of the first mutation linked to familial hypertrophic cardiomyopathy (HCM) an astonishing number of mutations affecting numerous sarcomeric proteins have been described. Among the most prevalent of these are mutations that affect thick filament binding proteins including the myosin essential and regulatory light chains and cardiac myosin binding protein-C (cMyBP-C). However, despite the frequency with which myosin binding proteins, especially cMyBP-C, have been linked to inherited cardiomyopathies, the functional consequences of mutations in these proteins and the mechanisms by which they cause disease are still only partly understood. The purpose of this review is to summarize the known disease-causing mutations that affect the major thick filament binding proteins and to relate these mutations to protein function. Conclusions emphasize the impact that discovery of HCM causing mutations has had on fueling insights into the basic biology of thick filament proteins and reinforce the idea that myosin binding proteins are dynamic regulators of the activation state of the thick filament that contribute to the speed and force of myosin driven muscle contraction. Additional work is still needed to determine the mechanisms by which individual mutations induce hypertrophic phenotypes. PMID:21415409
-
2015-01-01
Here, we present a universal, simple, efficient, and reliable way to add small BioBrick parts to any BioBrick via PCR that is compatible with BioBrick assembly standard 10. As a proof of principle, we have designed a universal primer, rbs_B0034, that contains a ribosomal binding site (RBS; BBa_B0034) and that can be used in PCR to amplify any coding BioBrick that starts with ATG. We performed test PCRs with rbs_B0034 on 31 different targets and found it to be 93.6% efficient. Moreover, when supplemented with a complementary primer, addition of RBS can be accomplished via whole plasmid site-directed mutagenesis, thus reducing the time required for further assembly of composite parts. The described method brings simplicity to the addition of small parts, such as regulatory elements to existing BioBricks. The final product of the PCR assembly is indistinguishable from the standard or 3A BioBrick assembly. PMID:25524097
-
DOE Office of Scientific and Technical Information (OSTI.GOV)
Strizhkov, B. N.; Drobyshev, A. L.; Mikhailovich, V. M.
PCR amplification on a microarray of gel-immobilized primers (microchip) has been developed. One of a pair of PCR primers was immobilized inside a separate microchip polyacrylamide porous gel pad of 0.1 x 0.1 x 0.02 (or 0.04) micron in size and 0.2 (or 0.4) nL in volume. The amplification was carried out simultaneously both in solution covering the microchip array and inside gel pads. Each gel pad contained the immobilized forward primers, while the fluorescently labeled reverse primers, as well as all components of the amplification reaction, diffused into the gel pads from the solution. To increase the amplification efficiency,more » the forward primers were also added into the solution. The kinetics of amplification was measured in real time in parallel for all gel pads with a fluorescent microscope equipped with a charge-coupled device (CCD) camera. The accuracy of the amplification was assessed by using the melting curves obtained for the duplexes formed by the labeled amplification product and the gel-immobilized primers during the amplification process; alternatively, the duplexes were produced by hybridization of the extended immobilized primers with labeled oligonucleotide probes. The on-chip amplification was applied to detect the anthrax toxin genes and the plasmid-borne beta-lactamase gene responsible for bacterial ampicillin resistance. The allele-specific type of PCR amplification was used to identify the Shiga toxin gene and discriminate it from the Shiga-like one. The genomic mutations responsible for rifampicin resistance of the Mycobacterium tuberculosis strains were detected by the same type of PCR amplification of the rpoB gene fragment isolated from sputum of tuberculosis patients. The on-chip PCR amplification has been shown to be a rapid, inexpensive and powerful tool to test genes responsible for bacterial toxin production and drug resistance, as well as to reveal point nucleotide mutations.« less
-
indCAPS: A tool for designing screening primers for CRISPR/Cas9 mutagenesis events.
Hodgens, Charles; Nimchuk, Zachary L; Kieber, Joseph J
2017-01-01
Genetic manipulation of organisms using CRISPR/Cas9 technology generally produces small insertions/deletions (indels) that can be difficult to detect. Here, we describe a technique to easily and rapidly identify such indels. Sequence-identified mutations that alter a restriction enzyme recognition site can be readily distinguished from wild-type alleles using a cleaved amplified polymorphic sequence (CAPS) technique. If a restriction site is created or altered by the mutation such that only one allele contains the restriction site, a polymerase chain reaction (PCR) followed by a restriction digest can be used to distinguish the two alleles. However, in the case of most CRISPR-induced alleles, no such restriction sites are present in the target sequences. In this case, a derived CAPS (dCAPS) approach can be used in which mismatches are purposefully introduced in the oligonucleotide primers to create a restriction site in one, but not both, of the amplified templates. Web-based tools exist to aid dCAPS primer design, but when supplied sequences that include indels, the current tools often fail to suggest appropriate primers. Here, we report the development of a Python-based, species-agnostic web tool, called indCAPS, suitable for the design of PCR primers used in dCAPS assays that is compatible with indels. This tool should have wide utility for screening editing events following CRISPR/Cas9 mutagenesis as well as for identifying specific editing events in a pool of CRISPR-mediated mutagenesis events. This tool was field-tested in a CRISPR mutagenesis experiment targeting a cytokinin receptor (AHK3) in Arabidopsis thaliana. The tool suggested primers that successfully distinguished between wild-type and edited alleles of a target locus and facilitated the isolation of two novel ahk3 null alleles. Users can access indCAPS and design PCR primers to employ dCAPS to identify CRISPR/Cas9 alleles at http://indcaps.kieber.cloudapps.unc.edu/.
-
Deep-sea vent phage DNA polymerase specifically initiates DNA synthesis in the absence of primers.
Zhu, Bin; Wang, Longfei; Mitsunobu, Hitoshi; Lu, Xueling; Hernandez, Alfredo J; Yoshida-Takashima, Yukari; Nunoura, Takuro; Tabor, Stanley; Richardson, Charles C
2017-03-21
A DNA polymerase is encoded by the deep-sea vent phage NrS-1. NrS-1 has a unique genome organization containing genes that are predicted to encode a helicase and a single-stranded DNA (ssDNA)-binding protein. The gene for an unknown protein shares weak homology with the bifunctional primase-polymerases (prim-pols) from archaeal plasmids but is missing the zinc-binding domain typically found in primases. We show that this gene product has efficient DNA polymerase activity and is processive in DNA synthesis in the presence of the NrS-1 helicase and ssDNA-binding protein. Remarkably, this NrS-1 DNA polymerase initiates DNA synthesis from a specific template DNA sequence in the absence of any primer. The de novo DNA polymerase activity resides in the N-terminal domain of the protein, whereas the C-terminal domain enhances DNA binding.
-
Bioinformatic Analysis of the Contribution of Primer Sequences to Aptamer Structures
Ellington, Andrew D.
2009-01-01
Aptamers are nucleic acid molecules selected in vitro to bind a particular ligand. While numerous experimental studies have examined the sequences, structures, and functions of individual aptamers, considerably fewer studies have applied bioinformatics approaches to try to infer more general principles from these individual studies. We have used a large Aptamer Database to parse the contributions of both random and constant regions to the secondary structures of more than 2000 aptamers. We find that the constant, primer-binding regions do not, in general, contribute significantly to aptamer structures. These results suggest that (a) binding function is not contributed to nor constrained by constant regions; (b) in consequence, the landscape of functional binding sequences is sparse but robust, favoring scenarios for short, functional nucleic acid sequences near origins; and (c) many pool designs for the selection of aptamers are likely to prove robust. PMID:18594898
-
Zhu, Jing; Ding, Yongshun; Liu, Xingti; Wang, Lei; Jiang, Wei
2014-09-15
Highly sensitive and selective detection strategy for single-base mutations is essential for risk assessment of malignancy and disease prognosis. In this work, a fluorescent detection method for single-base mutation was proposed based on high selectivity of toehold-mediated strand displacement reaction (TSDR) and powerful signal amplification capability of isothermal DNA amplification. A discrimination probe was specially designed with a stem-loop structure and an overhanging toehold domain. Hybridization between the toehold domain and the perfect matched target initiated the TSDR along with the unfolding of the discrimination probe. Subsequently, the target sequence acted as a primer to initiate the polymerization and nicking reactions, which released a great abundant of short sequences. Finally, the released strands were annealed with the reporter probe, launching another polymerization and nicking reaction to produce lots of G-quadruplex DNA, which could bind the N-methyl mesoporphyrin IX to yield an enhanced fluorescence response. However, when there was even a single base mismatch in the target DNA, the TSDR was suppressed and so subsequent isothermal DNA amplification and fluorescence response process could not occur. The proposed approach has been successfully implemented for the identification of the single-base mutant sequences in the human KRAS gene with a detection limit of 1.8 pM. Furthermore, a recovery of 90% was obtained when detecting the target sequence in spiked HeLa cells lysate, demonstrating the feasibility of this detection strategy for single-base mutations in biological samples. Copyright © 2014 Elsevier B.V. All rights reserved.
-
Deletion of transcription factor binding motifs using the CRISPR/spCas9 system in the β-globin LCR.
Kim, Yea Woon; Kim, AeRi
2017-07-20
Transcription factors play roles in gene transcription through direct binding to their motifs in genome, and inhibiting this binding provides an effective strategy for studying their roles. Here we applied the CRISPR/spCas9 system to mutate the binding motifs of transcription factors. Binding motifs for erythroid specific transcription factors were mutated in the locus control region hypersensitive sites of the human β-globin locus. Guide RNAs targeting binding motifs were cloned into lentiviral CRISPR vector containing the spCas9 gene, and transduced into MEL/ch11 cells carrying a human chromosome 11. DNA mutations in clonal cells were initially screened by quantitative PCR in genomic DNA and then clarified by sequencing. Mutations in binding motifs reduced occupancy by transcription factors in a chromatin environment. Characterization of mutations revealed that the CRISPR/spCas9 system mainly induced deletions in short regions of <20 bp and preferentially deleted nucleotides around the fifth nucleotide upstream of Protospacer adjacent motifs. These results indicate that the CRISPR/Cas9 system is suitable for mutating the binding motifs of transcription factors, and, consequently, would contribute to elucidate the direct roles of transcription factors. ©2017 The Author(s).
-
Leder, Verena; Lummer, Martina; Tegeler, Kathrin; Humpert, Fabian; Lewinski, Martin; Schüttpelz, Mark; Staiger, Dorothee
2014-10-10
Arabidopsis thaliana glycine-rich RNA binding protein 7 (AtGRP7) is part of a negative feedback loop through which it regulates alternative splicing and steady-state abundance of its pre-mRNA. Here we use fluorescence correlation spectroscopy to investigate the requirements for AtGRP7 binding to its intron using fluorescently-labelled synthetic oligonucleotides. By systematically introducing point mutations we identify three nucleotides that lead to an increased Kd value when mutated and thus are critical for AtGRP7 binding. Simultaneous mutation of all three residues abrogates binding. The paralogue AtGRP8 binds to an overlapping motif but with a different sequence preference, in line with overlapping but not identical functions of this protein pair. Truncation of the glycine-rich domain reduces the binding affinity of AtGRP7, showing for the first time that the glycine-rich stretch of a plant hnRNP-like protein contributes to binding. Mutation of the conserved R(49) that is crucial for AtGRP7 function in pathogen defence and splicing abolishes binding. Copyright © 2014 Elsevier Inc. All rights reserved.
-
Recombination of polynucleotide sequences using random or defined primers
Arnold, Frances H.; Shao, Zhixin; Affholter, Joseph A.; Zhao, Huimin H; Giver, Lorraine J.
2000-01-01
A method for in vitro mutagenesis and recombination of polynucleotide sequences based on polymerase-catalyzed extension of primer oligonucleotides is disclosed. The method involves priming template polynucleotide(s) with random-sequences or defined-sequence primers to generate a pool of short DNA fragments with a low level of point mutations. The DNA fragments are subjected to denaturization followed by annealing and further enzyme-catalyzed DNA polymerization. This procedure is repeated a sufficient number of times to produce full-length genes which comprise mutants of the original template polynucleotides. These genes can be further amplified by the polymerase chain reaction and cloned into a vector for expression of the encoded proteins.
-
Recombination of polynucleotide sequences using random or defined primers
Arnold, Frances H.; Shao, Zhixin; Affholter, Joseph A.; Zhao, Huimin; Giver, Lorraine J.
2001-01-01
A method for in vitro mutagenesis and recombination of polynucleotide sequences based on polymerase-catalyzed extension of primer oligonucleotides is disclosed. The method involves priming template polynucleotide(s) with random-sequences or defined-sequence primers to generate a pool of short DNA fragments with a low level of point mutations. The DNA fragments are subjected to denaturization followed by annealing and further enzyme-catalyzed DNA polymerization. This procedure is repeated a sufficient number of times to produce full-length genes which comprise mutants of the original template polynucleotides. These genes can be further amplified by the polymerase chain reaction and cloned into a vector for expression of the encoded proteins.
-
2016-01-01
Avoiding complementarity between primers when designing a PCR assay constitutes a central rule strongly anchored in the mind of the molecular scientist. 3’-complementarity will extend the primers during PCR elongation using one another as template, consequently disabling further possible involvement in traditional target amplification. However, a 5’-complementarity will leave the primers unchanged during PCR cycles, albeit sequestered to one another, therefore also suppressing target amplification. We show that 5’-complementarity between primers may be exploited in a new PCR method called COMplementary-Primer-Asymmetric (COMPAS)-PCR, using asymmetric primer concentrations to achieve target PCR amplification. Moreover, such a design may paradoxically reduce spurious non-target amplification by actively sequestering the limiting primer. The general principles were demonstrated using 5S rDNA direct repeats as target sequences to design a species-specific assay for identifying Salmo salar and Salmo trutta using almost fully complementary primers overlapping the same target sequence. Specificity was enhanced by using 3’-penultimate point mutations and the assay was further developed to enable identification of S. salar x S. trutta hybrids by High Resolution Melt analysis in a 35 min one-tube assay. This small paradigm shift, using highly complementary primers for PCR, should help develop robust assays that previously would not be considered. PMID:27783658
-
Poly(A) polymerase contains multiple functional domains.
Raabe, T; Murthy, K G; Manley, J L
1994-01-01
Poly(A) polymerase (PAP) contains regions of similarity with several known protein domains. Through site-directed mutagenesis, we provide evidence that PAP contains a functional ribonucleoprotein-type RNA binding domain (RBD) that is responsible for primer binding, making it the only known polymerase to contain such a domain. The RBD is adjacent to, and probably overlaps with, an apparent catalytic region responsible for polymerization. Despite the presence of sequence similarities, this catalytic domain appears to be distinct from the conserved polymerase module found in a large number of RNA-dependent polymerases. PAP contains two nuclear localization signals (NLSs) in its C terminus, each by itself similar to the consensus bipartite NLS found in many nuclear proteins. Mutagenesis experiments indicate that both signals, which are separated by nearly 140 residues, play important roles in directing PAP exclusively to the nucleus. Surprisingly, basic amino acids in the N-terminal-most NLS are also essential for AAUAAA-dependent polyadenylation but not for nonspecific poly(A) synthesis, suggesting that this region of PAP is involved in interactions both with nuclear targeting proteins and with nuclear polyadenylation factors. The serine/threonine-rich C terminus is multiply phosphorylated, including at sites affected by mutations in either NLS. Images PMID:8164653
-
Plant height revertants of Dominant Semidwarf mutant rice created by low-energy ion irradiation
NASA Astrophysics Data System (ADS)
Liu, Binmei; Wu, Yuejin; Xu, Xue; Song, M.; Zhao, M.; Fu, X. D.
2008-04-01
Dominant Semidwarf mutant rice (Sdd) was obtained from its wild type (WT) by irradiation with a low-energy ion beam. Six tall revertants of Sdd were induced by irradiation. The revertants restored the plant height to that of WT plants. Investigation of the agronomic character and genetic analysis indicate that the revertants are similar to WT plants with putative different inherited mutations. The revertants were checked for DNA differences using the simple sequence repeat technique. Among 408 such primers used, only 2 primers detected mutation sites in the revertants, which provided the molecular evidence for the revertants induced from Sdd. This study indicates that ion irradiation may be used as a mutagen to create revertants for plant architecture studies and could be a new application.
-
Smola, Matthew J; Rice, Greggory M; Busan, Steven; Siegfried, Nathan A; Weeks, Kevin M
2015-11-01
Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistries exploit small electrophilic reagents that react with 2'-hydroxyl groups to interrogate RNA structure at single-nucleotide resolution. Mutational profiling (MaP) identifies modified residues by using reverse transcriptase to misread a SHAPE-modified nucleotide and then counting the resulting mutations by massively parallel sequencing. The SHAPE-MaP approach measures the structure of large and transcriptome-wide systems as accurately as can be done for simple model RNAs. This protocol describes the experimental steps, implemented over 3 d, that are required to perform SHAPE probing and to construct multiplexed SHAPE-MaP libraries suitable for deep sequencing. Automated processing of MaP sequencing data is accomplished using two software packages. ShapeMapper converts raw sequencing files into mutational profiles, creates SHAPE reactivity plots and provides useful troubleshooting information. SuperFold uses these data to model RNA secondary structures, identify regions with well-defined structures and visualize probable and alternative helices, often in under 1 d. SHAPE-MaP can be used to make nucleotide-resolution biophysical measurements of individual RNA motifs, rare components of complex RNA ensembles and entire transcriptomes.
-
Grachev, M A; Zaychikov, E F; Ivanova, E M; Komarova, N I; Kutyavin, I V; Sidelnikova, N P; Frolova, I P
1984-01-01
Primer-dependent transcription by E. coli RNA polymerase on T7 promoter A2 has been studied. Synthetic deoxyribonucleotides complementary to the promoter over the region -8...+2 were taken as primers. A ribonucleoside residue was present at the 3'-end of some of these oligonucleotides. The octanucleotide complementary to the region -8...-1 appeared to be an active primer. Oligonucleotides having lengths from 3 to 6 nucleotide residues complementary to the promoter over the region -4...+2 also exhibited primer activity. The latter was some 5-10 times greater in the case of oligonucleotides having a ribonucleoside residue at the 3'-end. Oligonucleotides which on complementary binding do not reach the center of phosphodiester bond synthesis, as well as the decanucleotides (-8...+2) and octanucleotides (-6...+2) of both the ribo- and deoxyribo-series were inactive as primers. Images PMID:6390344
-
McCutchen-Maloney, Sandra L.
2002-01-01
Chimeric proteins having both DNA mutation binding activity and nuclease activity are synthesized by recombinant technology. The proteins are of the general formula A-L-B and B-L-A where A is a peptide having DNA mutation binding activity, L is a linker and B is a peptide having nuclease activity. The chimeric proteins are useful for detection and identification of DNA sequence variations including DNA mutations (including DNA damage and mismatches) by binding to the DNA mutation and cutting the DNA once the DNA mutation is detected.
-
Gumucio, D L; Rood, K L; Gray, T A; Riordan, M F; Sartor, C I; Collins, F S
1988-01-01
The molecular mechanisms responsible for the human fetal-to-adult hemoglobin switch have not yet been elucidated. Point mutations identified in the promoter regions of gamma-globin genes from individuals with nondeletion hereditary persistence of fetal hemoglobin (HPFH) may mark cis-acting sequences important for this switch, and the trans-acting factors which interact with these sequences may be integral parts in the puzzle of gamma-globin gene regulation. We have used gel retardation and footprinting strategies to define nuclear proteins which bind to the normal gamma-globin promoter and to determine the effect of HPFH mutations on the binding of a subset of these proteins. We have identified five proteins in human erythroleukemia cells (K562 and HEL) which bind to the proximal promoter region of the normal gamma-globin gene. One factor, gamma CAAT, binds the duplicated CCAAT box sequences; the -117 HPFH mutation increases the affinity of interaction between gamma CAAT and its cognate site. Two proteins, gamma CAC1 and gamma CAC2, bind the CACCC sequence. These proteins require divalent cations for binding. The -175 HPFH mutation interferes with the binding of a fourth protein, gamma OBP, which binds an octamer sequence (ATGCAAAT) in the normal gamma-globin promoter. The HPFH phenotype of the -175 mutation indicates that the octamer-binding protein may play a negative regulatory role in this setting. A fifth protein, EF gamma a, binds to sequences which overlap the octamer-binding site. The erythroid-specific distribution of EF gamma a and its close approximation to an apparent repressor-binding site suggest that it may be important in gamma-globin regulation. Images PMID:2468996
-
Design and cloning strategies for constructing shRNA expression vectors
McIntyre, Glen J; Fanning, Gregory C
2006-01-01
Background Short hairpin RNA (shRNA) encoded within an expression vector has proven an effective means of harnessing the RNA interference (RNAi) pathway in mammalian cells. A survey of the literature revealed that shRNA vector construction can be hindered by high mutation rates and the ensuing sequencing is often problematic. Current options for constructing shRNA vectors include the use of annealed complementary oligonucleotides (74 % of surveyed studies), a PCR approach using hairpin containing primers (22 %) and primer extension of hairpin templates (4 %). Results We considered primer extension the most attractive method in terms of cost. However, in initial experiments we encountered a mutation frequency of 50 % compared to a reported 20 – 40 % for other strategies. By modifying the technique to be an isothermal reaction using the DNA polymerase Phi29, we reduced the error rate to 10 %, making primer extension the most efficient and cost-effective approach tested. We also found that inclusion of a restriction site in the loop could be exploited for confirming construct integrity by automated sequencing, while maintaining intended gene suppression. Conclusion In this study we detail simple improvements for constructing and sequencing shRNA that overcome current limitations. We also compare the advantages of our solutions against proposed alternatives. Our technical modifications will be of tangible benefit to researchers looking for a more efficient and reliable shRNA construction process. PMID:16396676
-
Goodman, Stephen I; Binard, Robert J; Woontner, Michael R; Frerman, Frank E
2002-01-01
Glutaric acidemia type II is a human inborn error of metabolism which can be due to defects in either subunit of electron transfer flavoprotein (ETF) or in ETF:ubiquinone oxidoreductase (ETF:QO), but few disease-causing mutations have been described. The ETF:QO gene is located on 4q33, and contains 13 exons. Primers to amplify these exons are presented, together with mutations identified by molecular analysis of 20 ETF:QO-deficient patients. Twenty-one different disease-causing mutations were identified on 36 of the 40 chromosomes.
-
Taggart, David J.; Dayeh, Daniel M.; Fredrickson, Saul W.; Suo, Zucai
2014-01-01
The X-family DNA polymerases λ (Polλ) and β (Polβ) possess similar 5′-2-deoxyribose-5-phosphatelyase (dRPase) and polymerase domains. Besides these domains, Polλ also possesses a BRCA1 C-terminal (BRCT) domain and a proline-rich domain at its N terminus. However, it is unclear how these non-enzymatic domains contribute to the unique biological functions of Polλ. Here, we used primer extension assays and a newly developed high-throughput short oligonucleotide sequencing assay (HT-SOSA) to compare the efficiency of lesion bypass and fidelity of human Polβ, Polλ and two N-terminal deletion constructs of Polλ during the bypass of either an abasic site or a 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) lesion. We demonstrate that the BRCT domain of Polλ enhances the efficiency of abasic site bypass by approximately 1.6-fold. In contrast, deletion of the N-terminal domains of Polλ did not affect the efficiency of 8-oxodG bypass relative to nucleotide incorporations opposite undamaged dG. HT-SOSA analysis demonstrated that Polλ and Polβ preferentially generated −1 or −2 frameshift mutations when bypassing an abasic site and the single or double base deletion frequency was highly sequence dependent. Interestingly, the BRCT and proline-rich domains of Polλ cooperatively promoted the generation of −2 frameshift mutations when the abasic site was situated within a sequence context that was susceptible to homology-driven primer realignment. Furthermore, both N-terminal domains of Polλ increased the generation of −1 frameshift mutations during 8-oxodG bypass and influenced the frequency of substitution mutations produced by Polλ opposite the 8-oxodG lesion. Overall, our data support a model wherein the BRCT and proline-rich domains of Polλ act cooperatively to promote primer/template realignment between DNA strands of limited sequence homology. This function of the N-terminal domains may facilitate the role of Polλ as a gap-filling polymerase within the non-homologous end joining pathway. PMID:25108835
-
Xue, Jian; Wu, Riga; Pan, Yajiao; Wang, Shunxia; Qu, Baowang; Qin, Ying; Shi, Yuequn; Zhang, Chuchu; Li, Ran; Zhang, Liyan; Zhou, Cheng; Sun, Hongyu
2018-04-02
Massively parallel sequencing (MPS) technologies, also termed as next-generation sequencing (NGS), are becoming increasingly popular in study of short tandem repeats (STR). However, current library preparation methods are usually based on ligation or two-round PCR that requires more steps, making it time-consuming (about 2 days), laborious and expensive. In this study, a 16-plex STR typing system was designed with fusion primer strategy based on the Ion Torrent S5 XL platform which could effectively resolve the above challenges for forensic DNA database-type samples (bloodstains, saliva stains, etc.). The efficiency of this system was tested in 253 Han Chinese participants. The libraries were prepared without DNA isolation and adapter ligation, and the whole process only required approximately 5 h. The proportion of thoroughly genotyped samples in which all the 16 loci were successfully genotyped was 86% (220/256). Of the samples, 99.7% showed 100% concordance between NGS-based STR typing and capillary electrophoresis (CE)-based STR typing. The inconsistency might have been caused by off-ladder alleles and mutations in primer binding sites. Overall, this panel enabled the large-scale genotyping of the DNA samples with controlled quality and quantity because it is a simple, operation-friendly process flow that saves labor, time and costs. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
-
Walsh, Ann W.; Langley, David R.; Colonno, Richard J.; Tenney, Daniel J.
2010-01-01
Background Entecavir (ETV) is a deoxyguanosine analog competitive inhibitor of hepatitis B virus (HBV) polymerase that exhibits delayed chain termination of HBV DNA. A high barrier to entecavir-resistance (ETVr) is observed clinically, likely due to its potency and a requirement for multiple resistance changes to overcome suppression. Changes in the HBV polymerase reverse-transcriptase (RT) domain involve lamivudine-resistance (LVDr) substitutions in the conserved YMDD motif (M204V/I ± L180M), plus an additional ETV-specific change at residues T184, S202 or M250. These substitutions surround the putative dNTP binding site or primer grip regions of the HBV RT. Methods/Principal Findings To determine the mechanistic basis for ETVr, wildtype, lamivudine-resistant (M204V, L180M) and ETVr HBVs were studied using in vitro RT enzyme and cell culture assays, as well as molecular modeling. Resistance substitutions significantly reduced ETV incorporation and chain termination in HBV DNA and increased the ETV-TP inhibition constant (Ki) for HBV RT. Resistant HBVs exhibited impaired replication in culture and reduced enzyme activity (kcat) in vitro. Molecular modeling of the HBV RT suggested that ETVr residue T184 was adjacent to and stabilized S202 within the LVDr YMDD loop. ETVr arose through steric changes at T184 or S202 or by disruption of hydrogen-bonding between the two, both of which repositioned the loop and reduced the ETV-triphosphate (ETV-TP) binding pocket. In contrast to T184 and S202 changes, ETVr at primer grip residue M250 was observed during RNA-directed DNA synthesis only. Experimentally, M250 changes also impacted the dNTP-binding site. Modeling suggested a novel mechanism for M250 resistance, whereby repositioning of the primer-template component of the dNTP-binding site shifted the ETV-TP binding pocket. No structural data are available to confirm the HBV RT modeling, however, results were consistent with phenotypic analysis of comprehensive substitutions of each ETVr position. Conclusions Altogether, ETVr occurred through exclusion of ETV-TP from the dNTP-binding site, through different, novel mechanisms that involved lamivudine-resistance, ETV-specific substitutions, and the primer-template. PMID:20169198
-
Walsh, Ann W; Langley, David R; Colonno, Richard J; Tenney, Daniel J
2010-02-12
Entecavir (ETV) is a deoxyguanosine analog competitive inhibitor of hepatitis B virus (HBV) polymerase that exhibits delayed chain termination of HBV DNA. A high barrier to entecavir-resistance (ETVr) is observed clinically, likely due to its potency and a requirement for multiple resistance changes to overcome suppression. Changes in the HBV polymerase reverse-transcriptase (RT) domain involve lamivudine-resistance (LVDr) substitutions in the conserved YMDD motif (M204V/I +/- L180M), plus an additional ETV-specific change at residues T184, S202 or M250. These substitutions surround the putative dNTP binding site or primer grip regions of the HBV RT. To determine the mechanistic basis for ETVr, wildtype, lamivudine-resistant (M204V, L180M) and ETVr HBVs were studied using in vitro RT enzyme and cell culture assays, as well as molecular modeling. Resistance substitutions significantly reduced ETV incorporation and chain termination in HBV DNA and increased the ETV-TP inhibition constant (K(i)) for HBV RT. Resistant HBVs exhibited impaired replication in culture and reduced enzyme activity (k(cat)) in vitro. Molecular modeling of the HBV RT suggested that ETVr residue T184 was adjacent to and stabilized S202 within the LVDr YMDD loop. ETVr arose through steric changes at T184 or S202 or by disruption of hydrogen-bonding between the two, both of which repositioned the loop and reduced the ETV-triphosphate (ETV-TP) binding pocket. In contrast to T184 and S202 changes, ETVr at primer grip residue M250 was observed during RNA-directed DNA synthesis only. Experimentally, M250 changes also impacted the dNTP-binding site. Modeling suggested a novel mechanism for M250 resistance, whereby repositioning of the primer-template component of the dNTP-binding site shifted the ETV-TP binding pocket. No structural data are available to confirm the HBV RT modeling, however, results were consistent with phenotypic analysis of comprehensive substitutions of each ETVr position. Altogether, ETVr occurred through exclusion of ETV-TP from the dNTP-binding site, through different, novel mechanisms that involved lamivudine-resistance, ETV-specific substitutions, and the primer-template.
-
Romi, Erez; Baran, Nava; Gantman, Marina; Shmoish, Michael; Min, Bosun; Collins, Kathleen; Manor, Haim
2007-05-22
Telomerase is a cellular reverse transcriptase, which utilizes an integral RNA template to extend single-stranded telomeric DNA. We used site-specific photocrosslinking to map interactions between DNA primers and the catalytic protein subunit (tTERT) of Tetrahymena thermophila telomerase in functional enzyme complexes. Our assays reveal contact of the single-stranded DNA adjacent to the primer-template hybrid and tTERT residue W187 at the periphery of the N-terminal domain. This contact was detected in complexes with three different registers of template in the active site, suggesting that it is maintained throughout synthesis of a complete telomeric repeat. Substitution of nearby residue Q168, but not W187, alters the K(m) for primer elongation, implying that it plays a role in the DNA recognition. These findings are the first to directly demonstrate the physical location of TERT-DNA contacts in catalytically active telomerase and to identify amino acid determinants of DNA binding affinity. Our data also suggest a movement of the TERT active site relative to the template-adjacent single-stranded DNA binding site within a cycle of repeat synthesis.
-
Dong, Chongmei; Vincent, Kate; Sharp, Peter
2009-12-04
TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful tool for reverse genetics, combining traditional chemical mutagenesis with high-throughput PCR-based mutation detection to discover induced mutations that alter protein function. The most popular mutation detection method for TILLING is a mismatch cleavage assay using the endonuclease CelI. For this method, locus-specific PCR is essential. Most wheat genes are present as three similar sequences with high homology in exons and low homology in introns. Locus-specific primers can usually be designed in introns. However, it is sometimes difficult to design locus-specific PCR primers in a conserved region with high homology among the three homoeologous genes, or in a gene lacking introns, or if information on introns is not available. Here we describe a mutation detection method which combines High Resolution Melting (HRM) analysis of mixed PCR amplicons containing three homoeologous gene fragments and sequence analysis using Mutation Surveyor software, aimed at simultaneous detection of mutations in three homoeologous genes. We demonstrate that High Resolution Melting (HRM) analysis can be used in mutation scans in mixed PCR amplicons containing three homoeologous gene fragments. Combining HRM scanning with sequence analysis using Mutation Surveyor is sensitive enough to detect a single nucleotide mutation in the heterozygous state in a mixed PCR amplicon containing three homoeoloci. The method was tested and validated in an EMS (ethylmethane sulfonate)-treated wheat TILLING population, screening mutations in the carboxyl terminal domain of the Starch Synthase II (SSII) gene. Selected identified mutations of interest can be further analysed by cloning to confirm the mutation and determine the genomic origin of the mutation. Polyploidy is common in plants. Conserved regions of a gene often represent functional domains and have high sequence similarity between homoeologous loci. The method described here is a useful alternative to locus-specific based methods for screening mutations in conserved functional domains of homoeologous genes. This method can also be used for SNP (single nucleotide polymorphism) marker development and eco-TILLING in polyploid species.
-
Hashimoto, Masahiko; Barany, Francis; Xu, Feng; Soper, Steven A
2007-09-01
We have fabricated a flow-through biochip consisting of passive elements for the analysis of single base mutations in genomic DNA using polycarbonate (PC) as the substrate. The biochip was configured to carry out two processing steps on the input sample, a primary polymerase chain reaction (PCR) followed by an allele-specific ligation detection reaction (LDR) for scoring the presence of low abundant point mutations in genomic DNA. The operation of the device was demonstrated by detecting single nucleotide polymorphisms in gene fragments (K-ras) that carry high diagnostic value for colorectal cancers. The effect of carryover from the primary PCR on the subsequent LDR was investigated in terms of LDR yield and fidelity. We found that a post-PCR treatment step prior to the LDR phase of the assay was not essential. As a consequence, a thermal cycling microchip was used for a sequential PCR/LDR in a simple continuous-flow format, in which the following three steps were carried out: (1) exponential amplification of the gene fragments from genomic DNA; (2) mixing of the resultant PCR product(s) with an LDR cocktail via a Y-shaped passive micromixer; and (3) ligation of two primers (discriminating primer that carried the complement base to the mutation locus being interrogated and a common primer) only when the particular mutation was present in the genomic DNA. We successfully demonstrated the ability to detect one mutant DNA in 1000 normal sequences with the integrated microfluidic system. The PCR/LDR assay using the microchip performed the entire assay at a relatively fast processing speed: 18.7 min for 30 rounds of PCR, 4.1 min for 13 rounds of LDR (total processing time = ca. 22.8 min) and could screen multiple mutations simultaneously in a multiplexed format. In addition, the low cost of the biochip due to the fact that it was fabricated from polymers using replication technologies and consisted of passive elements makes the platform amenable to clinical diagnostics, where one-time use devices are required to eliminate false positives resulting from carryover contamination.
-
SAAMBE: Webserver to Predict the Charge of Binding Free Energy Caused by Amino Acids Mutations.
Petukh, Marharyta; Dai, Luogeng; Alexov, Emil
2016-04-12
Predicting the effect of amino acid substitutions on protein-protein affinity (typically evaluated via the change of protein binding free energy) is important for both understanding the disease-causing mechanism of missense mutations and guiding protein engineering. In addition, researchers are also interested in understanding which energy components are mostly affected by the mutation and how the mutation affects the overall structure of the corresponding protein. Here we report a webserver, the Single Amino Acid Mutation based change in Binding free Energy (SAAMBE) webserver, which addresses the demand for tools for predicting the change of protein binding free energy. SAAMBE is an easy to use webserver, which only requires that a coordinate file be inputted and the user is provided with various, but easy to navigate, options. The user specifies the mutation position, wild type residue and type of mutation to be made. The server predicts the binding free energy change, the changes of the corresponding energy components and provides the energy minimized 3D structure of the wild type and mutant proteins for download. The SAAMBE protocol performance was tested by benchmarking the predictions against over 1300 experimentally determined changes of binding free energy and a Pearson correlation coefficient of 0.62 was obtained. How the predictions can be used for discriminating disease-causing from harmless mutations is discussed. The webserver can be accessed via http://compbio.clemson.edu/saambe_webserver/.
-
Gabus, C; Ficheux, D; Rau, M; Keith, G; Sandmeyer, S; Darlix, J L
1998-01-01
Retroviruses, including HIV-1 and the distantly related yeast retroelement Ty3, all encode a nucleoprotein required for virion structure and replication. During an in vitro comparison of HIV-1 and Ty3 nucleoprotein function in RNA dimerization and cDNA synthesis, we discovered a bipartite primer-binding site (PBS) for Ty3 composed of sequences located at opposite ends of the genome. Ty3 cDNA synthesis requires the 3' PBS for primer tRNAiMet annealing to the genomic RNA, and the 5' PBS, in cis or in trans, as the reverse transcription start site. Ty3 RNA alone is unable to dimerize, but formation of dimeric tRNAiMet bound to the PBS was found to direct dimerization of Ty3 RNA-tRNAiMet. Interestingly, HIV-1 nucleocapsid protein NCp7 and Ty3 NCp9 were interchangeable using HIV-1 and Ty3 RNA template-primer systems. Our findings impact on the understanding of non-canonical reverse transcription as well as on the use of Ty3 systems to screen for anti-NCp7 drugs. PMID:9707446
-
Gabus, C; Ficheux, D; Rau, M; Keith, G; Sandmeyer, S; Darlix, J L
1998-08-17
Retroviruses, including HIV-1 and the distantly related yeast retroelement Ty3, all encode a nucleoprotein required for virion structure and replication. During an in vitro comparison of HIV-1 and Ty3 nucleoprotein function in RNA dimerization and cDNA synthesis, we discovered a bipartite primer-binding site (PBS) for Ty3 composed of sequences located at opposite ends of the genome. Ty3 cDNA synthesis requires the 3' PBS for primer tRNAiMet annealing to the genomic RNA, and the 5' PBS, in cis or in trans, as the reverse transcription start site. Ty3 RNA alone is unable to dimerize, but formation of dimeric tRNAiMet bound to the PBS was found to direct dimerization of Ty3 RNA-tRNAiMet. Interestingly, HIV-1 nucleocapsid protein NCp7 and Ty3 NCp9 were interchangeable using HIV-1 and Ty3 RNA template-primer systems. Our findings impact on the understanding of non-canonical reverse transcription as well as on the use of Ty3 systems to screen for anti-NCp7 drugs.
-
Muneta, Yoshihiro; Minagawa, Yu; Kusumoto, Masahiro; Shinkai, Hiroki; Uenishi, Hirohide; Splichal, Igor
2012-05-01
In the present study, we have developed an allele-specific primer-polymerase chain reaction (ASP-PCR) for genotyping a single nucleotide polymorphism (SNP) of swine Toll-like receptor 2 (TLR2) (C406G), which is related to the prevalence of pneumonia caused by Mycoplasma hyopneumoniae. We also compared the allele frequency among several pig breeds of Japan and the Czech Republic. Allele-specific primers were constructed by introducing 1-base mismatch sequence before the SNP site. The swine TLR2 C406G mutation was successfully determined by the ASP-PCR using genomic DNA samples in Japan as previously genotyped by a sequencing method. Using the PCR condition determined, genomic DNA samples from pig blood obtained from 110 pigs from 7 different breeds in the Czech Republic were genotyped by the ASP-PCR. The genotyping results from the ASP-PCR were completely matched with the results from the sequencing method. The allele frequency of the swine TLR2 C406G mutation was 27.5% in the Czech Republic and 3.6% in Japan. The C406G mutation was only found in the Landrace breed in Japan, and was almost exclusively found in the Landrace breed in the Czech Republic as well. These results indicated the usefulness of ASP-PCR for detecting a specific SNP for swine TLR2.
-
The Use of Aptamers in Breast Cancer Anti-Metastasis Therapy
2001-09-01
with serum-free medium (IRL-41) supplemented with 0.4 mM ascorbic acid and antibiotics for 72 h (2,3). Recombinant P4(108_343)-HA was purified from...mCLCA1 and hCLCA1 (9, calcium -activated chloride channel; cystic fibrosis 11). Although the protein processing and function ap- pear to be conserved among...AAC) to glutamine ( CAA ) mutation that abolishes a derived primers followed by internal gene-specific primers consensus glycosylation site (NxS/T-QxS/T
-
Molecular Analysis of Glucose-6-Phosphate Dehydrogenase Gene Mutations in Bangladeshi Individuals.
Sarker, Suprovath Kumar; Islam, Md Tarikul; Eckhoff, Grace; Hossain, Mohammad Amir; Qadri, Syeda Kashfi; Muraduzzaman, A K M; Bhuyan, Golam Sarower; Shahidullah, Mohammod; Mannan, Mohammad Abdul; Tahura, Sarabon; Hussain, Manzoor; Akhter, Shahida; Nahar, Nazmun; Shirin, Tahmina; Qadri, Firdausi; Mannoor, Kaiissar
2016-01-01
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common X-linked human enzyme defect of red blood cells (RBCs). Individuals with this gene defect appear normal until exposed to oxidative stress which induces hemolysis. Consumption of certain foods such as fava beans, legumes; infection with bacteria or virus; and use of certain drugs such as primaquine, sulfa drugs etc. may result in lysis of RBCs in G6PD deficient individuals. The genetic defect that causes G6PD deficiency has been identified mostly as single base missense mutations. One hundred and sixty G6PD gene mutations, which lead to amino acid substitutions, have been described worldwide. The purpose of this study was to detect G6PD gene mutations in hospital-based settings in the local population of Dhaka city, Bangladesh. Qualitative fluorescent spot test and quantitative enzyme activity measurement using RANDOX G6PDH kit were performed for analysis of blood specimens and detection of G6PD-deficient participants. For G6PD-deficient samples, PCR was done with six sets of primers specific for G6PD gene. Automated Sanger sequencing of the PCR products was performed to identify the mutations in the gene. Based on fluorescence spot test and quantitative enzyme assay followed by G6PD gene sequencing, 12 specimens (11 males and one female) among 121 clinically suspected patient-specimens were found to be deficient, suggesting a frequency of 9.9% G6PD deficiency. Sequencing of the G6PD-deficient samples revealed c.C131G substitution (exon-3: Ala44Gly) in six samples, c.G487A substitution (exon-6:Gly163Ser) in five samples and c.G949A substitution (exon-9: Glu317Lys) of coding sequence in one sample. These mutations either affect NADP binding or disrupt protein structure. From the study it appears that Ala44Gly and Gly163Ser are the most common G6PD mutations in Dhaka, Bangladesh. This is the first study of G6PD mutations in Bangladesh.
-
Molecular Analysis of Glucose-6-Phosphate Dehydrogenase Gene Mutations in Bangladeshi Individuals
Sarker, Suprovath Kumar; Hossain, Mohammad Amir; Qadri, Syeda Kashfi; Muraduzzaman, A. K. M.; Bhuyan, Golam Sarower; Shahidullah, Mohammod; Mannan, Mohammad Abdul; Tahura, Sarabon; Hussain, Manzoor; Akhter, Shahida; Nahar, Nazmun; Shirin, Tahmina; Qadri, Firdausi; Mannoor, Kaiissar
2016-01-01
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common X-linked human enzyme defect of red blood cells (RBCs). Individuals with this gene defect appear normal until exposed to oxidative stress which induces hemolysis. Consumption of certain foods such as fava beans, legumes; infection with bacteria or virus; and use of certain drugs such as primaquine, sulfa drugs etc. may result in lysis of RBCs in G6PD deficient individuals. The genetic defect that causes G6PD deficiency has been identified mostly as single base missense mutations. One hundred and sixty G6PD gene mutations, which lead to amino acid substitutions, have been described worldwide. The purpose of this study was to detect G6PD gene mutations in hospital-based settings in the local population of Dhaka city, Bangladesh. Qualitative fluorescent spot test and quantitative enzyme activity measurement using RANDOX G6PDH kit were performed for analysis of blood specimens and detection of G6PD-deficient participants. For G6PD-deficient samples, PCR was done with six sets of primers specific for G6PD gene. Automated Sanger sequencing of the PCR products was performed to identify the mutations in the gene. Based on fluorescence spot test and quantitative enzyme assay followed by G6PD gene sequencing, 12 specimens (11 males and one female) among 121 clinically suspected patient-specimens were found to be deficient, suggesting a frequency of 9.9% G6PD deficiency. Sequencing of the G6PD-deficient samples revealed c.C131G substitution (exon-3: Ala44Gly) in six samples, c.G487A substitution (exon-6:Gly163Ser) in five samples and c.G949A substitution (exon-9: Glu317Lys) of coding sequence in one sample. These mutations either affect NADP binding or disrupt protein structure. From the study it appears that Ala44Gly and Gly163Ser are the most common G6PD mutations in Dhaka, Bangladesh. This is the first study of G6PD mutations in Bangladesh. PMID:27880809
-
Ko, Chun Hay; Yung, Edmund; Li, Karen; Li, Chung Leung; Ng, Pak Cheung; Fung, Kwok Pui; Wong, Raymond Pui-On; Chui, Kit Man; Gu, Goldie Jia-Shi; Fok, Tai Fai
2006-01-01
The primary objective of our study was to provide a simple and reliable assay for identifying the majority of G6PD genetic variants in the Chinese population. We optimized the multiplex primer extension reaction (MPER) assay for simultaneous screening of 14-point mutations in 98 G6PD-deficient subjects. Our data demonstrated that this method is precise, cost-effective and has successfully identified mutations in 97 out of 98 subjects, including all heterozygous mutants. We also detected a relatively high incidence (12.3%) of c.871G > A, and all of them harbored the silent mutation c.1311C > T. Apart from the screening program, the pharmacogenetic relationship between G6PD level and residual reduced glutathione (GSH) level was studied upon oxidative challenge by alpha-naphthol. The GSH levels were correlated with their status of G6PD deficiency, but no significant difference was observed between individual G6PD-deficient groups. Our data demonstrated the potentials of the MPER assay for characterization of G6PD deficiency and other genetic diseases.
-
NASA Astrophysics Data System (ADS)
Ezerski, Jacob; Cheung, Margaret
CaM forms distinct conformation states through modifications in its charge distribution upon binding to Ca2+ ions. The occurrence of protein structural change resulting from an altered charge distribution is paramount in the scheme of cellular signaling. Not only is charge induced structural change observed in CaM, it is also seen in an essential binding target: calmodulin-depended protein kinase II (CaMKII). In order to investigate the mechanism of selectivity in relation to changes in secondary structure, the CaM binding domain of CaMKII is isolated. Experimentally, charged residues of the CaMKII peptide are systematically mutated to alanine, resulting in altered binding kinetics between the peptide and the Ca2+ saturated state of CaM. We perform an all atom simulation of the wildtype (RRK) and mutated (AAA) CaMKII peptides and generate structures from the trajectory. We analyze RRK and AAA using DSSP and find significant structural differences due to the mutation. Structures from the RRK and AAA ensembles are then selected and docked onto the crystal structure of Ca2+ saturated CaM. We observe that RRK binds to CaM at the C-terminus, whereas the 3-residue mutation, AAA, shows increased patterns of binding to the N-terminus and linker regions of CaM. Due to the conformational change of the peptide ensemble from charged residue mutation, a distinct change in the binding site can be seen, which offers an explanation to experimentally observed changes in kinetic binding rates
-
Xu, Li; Ji, Jin-Jun; Le, Wangping; Xu, Yan S; Dou, Dandan; Pan, Jieli; Jiao, Yifeng; Zhong, Tianfei; Wu, Dehong; Wang, Yumei; Wen, Chengping; Xie, Guan-Qun; Yao, Feng; Zhao, Heng; Fan, Yong-Sheng; Chin, Y Eugene
2015-10-15
Cytokine or growth factor activated STAT3 undergoes multiple post-translational modifications, dimerization and translocation into nuclei, where it binds to serum-inducible element (SIE, 'TTC(N3)GAA')-bearing promoters to activate transcription. The STAT3 DNA binding domain (DBD, 320-494) mutation in hyper immunoglobulin E syndrome (HIES), called the HIES mutation (R382Q, R382W or V463Δ), which elevates IgE synthesis, inhibits SIE binding activity and sensitizes genes such as TNF-α for expression. However, the mechanism by which the HIES mutation sensitizes STAT3 in gene induction remains elusive. Here, we report that STAT3 binds directly to the AGG-element with the consensus sequence 'AGG(N3)AGG'. Surprisingly, the helical N-terminal region (1-355), rather than the canonical STAT3 DBD, is responsible for AGG-element binding. The HIES mutation markedly enhances STAT3 AGG-element binding and AGG-promoter activation activity. Thus, STAT3 is a dual specificity transcription factor that promotes gene expression not only via SIE- but also AGG-promoter activity. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
-
Kibenge, Molly Jt; Iwamoto, Tokinori; Wang, Yingwei; Morton, Alexandra; Routledge, Richard; Kibenge, Frederick Sb
2016-01-06
Infectious salmon anaemia (ISA) virus (ISAV) belongs to the genus Isavirus, family Orthomyxoviridae. ISAV occurs in two basic genotypes, North American and European. The European genotype is more widespread and shows greater genetic variation and greater virulence variation than the North American genotype. To date, all of the ISAV isolates from the clinical disease, ISA, have had deletions in the highly polymorphic region (HPR) on ISAV segment 6 (ISAV-HPRΔ) relative to ISAV-HPR0, named numerically from ISAV-HPR1 to over ISAV-HPR30. ISA outbreaks have only been reported in farmed Atlantic salmon, although ISAV has been detected by RT-PCR in wild fish. It is recognized that asymptomatically ISAV-infected fish exist. There is no universally accepted ISAV RT-qPCR TaqMan® assay. Most diagnostic laboratories use the primer-probe set targeting a 104 bp-fragment on ISAV segment 8. Some laboratories and researchers have found a primer-probe set targeting ISAV segment 7 to be more sensitive. Other researchers have published different ISAV segment 8 primer-probe sets that are highly sensitive. In this study, we tested 1,106 fish tissue samples collected from (i) market-bought farmed salmonids and (ii) wild salmon from throughout British Columbia (BC), Canada, for ISAV using real time RT-qPCR targeting segment 8 and/or conventional RT-PCR with segment 8 primers and segment 6 HPR primers, and by virus isolation attempts using Salmon head kidney (SHK-1 and ASK-2) cell line monolayers. The sequences from the conventional PCR products were compared by multiple alignment and phylogenetic analyses. Seventy-nine samples were "non-negative" with at least one of these tests in one or more replicates. The ISAV segment 6 HPR sequences from the PCR products matched ISAV variants, HPR5 on 29 samples, one sample had both HPR5 and HPR7b and one matched HPR0. All sequences were of European genotype. In addition, alignment of sequences of the conventional PCR product segment 8 showed they had a single nucleotide mutation in the region of the probe sequence and a 9-nucleotide overlap with the reverse primer sequence of the real time RT-qPCR assay. None of the classical ISAV segment 8 sequences in the GenBank have this mutation in the probe-binding site of the assay, suggesting the presence of a novel ISAV variant in BC. A phylogenetic tree of these sequences showed that some ISAV sequences diverted early from the classical European genotype sequences, while others have evolved separately. All virus isolation attempts on the samples were negative, and thus the samples were considered "negative" in terms of the threshold trigger set for Canadian federal regulatory action; i.e., successful virus isolation in cell culture. This is the first published report of the detection of ISAV sequences in fish from British Columbia, Canada. The sequences detected, both of ISAV-HPRΔ and ISAV-HPR0 are of European genotype. These sequences are different from the classical ISAV segment 8 sequences, and this difference suggests the presence of a new ISAV variant of European genotype in BC. Our results further suggest that ISAV-HPRΔ strains can be present without clinical disease in farmed fish and without being detected by virus isolation using fish cell lines.
-
Cristofari, G; Gabus, C; Ficheux, D; Bona, M; Le Grice, S F; Darlix, J L
1999-12-17
Human immunodeficiency virus (HIV) and the distantly related yeast Ty3 retrotransposon encode reverse transcriptase (RT) and a nucleic acid-binding protein designated nucleocapsid protein (NCp) with either one or two zinc fingers, required for HIV-1 replication and Ty3 transposition, respectively. In vitro binding of HIV-1 NCp7 to viral 5' RNA and primer tRNA(3)(Lys) catalyzes formation of nucleoprotein complexes resembling the virion nucleocapsid. Nucleocapsid complex formation functions in viral RNA dimerization and tRNA annealing to the primer binding site (PBS). RT is recruited in these nucleoprotein complexes and synthesizes minus-strand cDNA initiated at the PBS. Recent results on yeast Ty3 have shown that the homologous NCp9 promotes annealing of primer tRNA(i)(Met) to a 5'-3' bipartite PBS, allowing RNA:tRNA dimer formation and initiation of cDNA synthesis at the 5' PBS (). To compare specific cDNA synthesis in a retrotransposon and HIV-1, we have established a Ty3 model system comprising Ty3 RNA with the 5'-3' PBS, primer tRNA(i)(Met), NCp9, and for the first time, highly purified Ty3 RT. Here we report that Ty3 RT is as active as retroviral HIV-1 or murine leukemia virus RT using a synthetic template-primer system. Moreover, and in contrast to what was found with retroviral RTs, retrotransposon Ty3 RT was unable to direct cDNA synthesis by self-priming. We also show that Ty3 nucleoprotein complexes were formed in vitro and that the N terminus of NCp9, but not the zinc finger, is required for complex formation, tRNA annealing to the PBS, RNA dimerization, and primer tRNA-directed cDNA synthesis by Ty3 RT. These results indicate that NCp9 chaperones bona fide cDNA synthesis by RT in the yeast Ty3 retrotransposon, as illustrated for NCp7 in HIV-1, reinforcing the notion that Ty3 NCp9 is an ancestor of HIV-1 NCp7.
-
Primer-Free Aptamer Selection Using A Random DNA Library
Pan, Weihua; Xin, Ping; Patrick, Susan; Dean, Stacey; Keating, Christine; Clawson, Gary
2010-01-01
Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified through an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX) to recognize a wide variety of targets, from small molecules to proteins and other macromolecules 2-4. Aptamers have properties that are well suited for in vivo diagnostic and/or therapeutic applications: Besides good specificity and affinity, they are easily synthesized, survive more rigorous processing conditions, they are poorly immunogenic, and their relatively small size can result in facile penetration of tissues. Aptamers that are identified through the standard SELEX process usually comprise ~80 nucleotides (nt), since they are typically selected from nucleic acid libraries with ~40 nt long randomized regions plus fixed primer sites of ~20 nt on each side. The fixed primer sequences thus can comprise nearly ~50% of the library sequences, and therefore may positively or negatively compromise identification of aptamers in the selection process 3, although bioinformatics approaches suggest that the fixed sequences do not contribute significantly to aptamer structure after selection 5. To address these potential problems, primer sequences have been blocked by complementary oligonucleotides or switched to different sequences midway during the rounds of SELEX 6, or they have been trimmed to 6-9 nt 7, 8. Wen and Gray 9 designed a primer-free genomic SELEX method, in which the primer sequences were completely removed from the library before selection and were then regenerated to allow amplification of the selected genomic fragments. However, to employ the technique, a unique genomic library has to be constructed, which possesses limited diversity, and regeneration after rounds of selection relies on a linear reamplification step. Alternatively, efforts to circumvent problems caused by fixed primer sequences using high efficiency partitioning are met with problems regarding PCR amplification 10. We have developed a primer-free (PF) selection method that significantly simplifies SELEX procedures and effectively eliminates primer-interference problems 11, 12. The protocols work in a straightforward manner. The central random region of the library is purified without extraneous flanking sequences and is bound to a suitable target (for example to a purified protein or complex mixtures such as cell lines). Then the bound sequences are obtained, reunited with flanking sequences, and re-amplified to generate selected sub-libraries. As an example, here we selected aptamers to S100B, a protein marker for melanoma. Binding assays showed Kd s in the 10-7 - 10-8 M range after a few rounds of selection, and we demonstrate that the aptamers function effectively in a sandwich binding format. PMID:20689511
-
NASA Astrophysics Data System (ADS)
Tsujimura, M.; Akutsu, J.; Zhang, Z.; Sasaki, M.; Tajima, H.; Kawarabayasi, Y.
2004-12-01
The thermostable proteins or enzymes were expected to be capable to be utilized in many areas of industries. Many thermophilic microorganisms, which possess the thermostable proteins or enzymes, were identified from the extreme environment. However, many unidentified and uncultivable microorganisms are still remaining in the environment on the earth. It is generally said that the cultivable microorganisms are less than 1% of entire microorganisms living in the earth, remaining over 99% are still uncultivable. As an approach to the uncultivable microorganisms, the PCR amplification of 16S rDNA region using primer sets designed from the conserved region has been generally utilized for detection and community analysis of microorganism in the environment. However, the facts, that PCR amplification introduces the mutation in the amplified DNA fragment and efficiency of PCR amplification is depend on the sequences of primer sets, indicated that the improving of PCR analysis was necessary for more correct detection of microorganisms. As the result of evaluation for the quality of DNA polymerases, sequences of primers used for amplification and conditions of PCR amplification, the DNA polymerase, the primer set and the conditions for amplification, which did not amplify the DNA fragment from the DNA contaminated within the DNA polymerase itself, were successfully selected. Also the rate of mutation in the DNA fragment amplified was evaluated using this conditions and the genomic DNA from cultivable microbes as a template. The result indicated the rate of mutation introduced by PCR was approximately 0.1% to 0.125%. The improved method using these conditions and error rate calculated was applied for the analysis of microorganisms in the geothermal environment. The result indicated that four kinds of dominant microorganisms, including both of bacteria and archaea, were alive within soil in the hot spring in Tohoku Area. We would like to apply this improved method to detection of microorganisms with important genes from more other environments.
-
Petukh, Marharyta; Li, Minghui; Alexov, Emil
2015-07-01
A new methodology termed Single Amino Acid Mutation based change in Binding free Energy (SAAMBE) was developed to predict the changes of the binding free energy caused by mutations. The method utilizes 3D structures of the corresponding protein-protein complexes and takes advantage of both approaches: sequence- and structure-based methods. The method has two components: a MM/PBSA-based component, and an additional set of statistical terms delivered from statistical investigation of physico-chemical properties of protein complexes. While the approach is rigid body approach and does not explicitly consider plausible conformational changes caused by the binding, the effect of conformational changes, including changes away from binding interface, on electrostatics are mimicked with amino acid specific dielectric constants. This provides significant improvement of SAAMBE predictions as indicated by better match against experimentally determined binding free energy changes over 1300 mutations in 43 proteins. The final benchmarking resulted in a very good agreement with experimental data (correlation coefficient 0.624) while the algorithm being fast enough to allow for large-scale calculations (the average time is less than a minute per mutation).
-
Maksay, Gábor; Bíró, Tímea; Laube, Bodo; Nemes, Péter
2008-01-01
Human alpha1 and hyperekplexia mutant alpha1(R271L) glycine receptors (GlyRs) were transiently expressed in human embryonic kidney 293 cells for [3H]strychnine binding. Binding parameters were determined using a ternary allosteric model. The hyperekplexia mutation increased the positive cooperativity of 0.3 mM propofol and glycine binding by about six times: the cooperativity factor beta was 0.26 for alpha1 GlyRs and 0.04 for alpha1(R271L) GlyRs. Thus, propofol restored the potency of glycine impaired by the mutation. Five nortropeines, i.e. substituted benzoates of nortropine and a new compound, nortropisetron were prepared and also examined on [3H]strychnine binding. They showed nanomolar displacing potencies amplified by the hyperekplexia mutation. The affinity of nor-O-zatosetron (2.6 nM) is one of the highest reported for GlyRs. This binding test offers an in vitro method to evaluate agents against neurological disorders associated with inherited mutations of GlyRs.
-
Yang, W C; Zhu, L; Zhou, B X; Tania, S; Zhou, Q; Khan, M A; Fu, X L; Cheng, J L; Lv, H B; Fu, J J
2015-09-25
Retinitis pigmentosa (RP) is a retinal degenerative disorder that often causes complete blindness. Mutations of more than 50 genes have been identified as associated with RP, including the CACNA1F gene. In a recent study, by employing next-generation sequencing, we identified a novel mutation in the CACNA1F gene. In this study, we used the amplification refractory mutation system (ARMS) and identified a single nucleotide change c.1555C>T in exon 13 of the CACNA1F gene, leading to the substitution of arginine by tryptophan (p.R519W) in a Chinese individual affected by RP. This study actually confirms this novel mutation, and establishes the ARMS technique for the detection of mutations in RP.
-
Zhu, Yanyan; Yuan, Yuan; Xiao, Xiuchan; Zhang, Liyun; Guo, Yanzhi; Pu, Xuemei
2014-11-01
G-protein-coupled receptors (GPCRs) are currently one of the largest families of drug targets. The constitutive activation induced by mutation of key GPCR residues is associated closely with various diseases. However, the structural basis underlying such activation and its role in drug binding has remained unclear. Herein, we used all-atom molecular dynamics simulations and free energy calculations to study the effects of a D130N mutation on the structure of β2 adrenergic receptor (β2AR) and its binding of the agonist salbutamol. The results indicate that the mutation caused significant changes in some key helices. In particular, the mutation leads to the departure of transmembrane 3 (TM3) from transmembrane 6 (TM6) and marked changes in the NPxxY region as well as the complete disruption of a key ionic lock, all of which contribute to the observed constitutive activation. In addition, the D130N mutation weakens some important H-bonds, leading to structural changes in these regions. Binding free energy calculations indicate that van der Waals and electrostatic interactions are the main driving forces in binding salbutamol; however, binding strength in the mutant β2AR is significantly enhanced mainly through modifying electrostatic interactions. Further analysis revealed that the increase in binding energy upon mutation stems mainly from the H-bonds formed between the hydroxyl group of salbutamol and the serine residues of TM5. This observation suggests that modifications of the H-bond groups of this drug could significantly influence drug efficacy in the treatment of diseases associated with this mutation.
-
Green, Stefan J.; Venkatramanan, Raghavee; Naqib, Ankur
2015-01-01
The polymerase chain reaction (PCR) is sensitive to mismatches between primer and template, and mismatches can lead to inefficient amplification of targeted regions of DNA template. In PCRs in which a degenerate primer pool is employed, each primer can behave differently. Therefore, inefficiencies due to different primer melting temperatures within a degenerate primer pool, in addition to mismatches between primer binding sites and primers, can lead to a distortion of the true relative abundance of targets in the original DNA pool. A theoretical analysis indicated that a combination of primer-template and primer-amplicon interactions during PCR cycles 3–12 is potentially responsible for this distortion. To test this hypothesis, we developed a novel amplification strategy, entitled “Polymerase-exonuclease (PEX) PCR”, in which primer-template interactions and primer-amplicon interactions are separated. The PEX PCR method substantially and significantly improved the evenness of recovery of sequences from a mock community of known composition, and allowed for amplification of templates with introduced mismatches near the 3’ end of the primer annealing sites. When the PEX PCR method was applied to genomic DNA extracted from complex environmental samples, a significant shift in the observed microbial community was detected. Furthermore, the PEX PCR method provides a mechanism to identify which primers in a primer pool are annealing to target gDNA. Primer utilization patterns revealed that at high annealing temperatures in the PEX PCR method, perfect match annealing predominates, while at lower annealing temperatures, primers with up to four mismatches with templates can contribute substantially to amplification. The PEX PCR method is simple to perform, is limited to PCR mixes and a single exonuclease step which can be performed without reaction cleanup, and is recommended for reactions in which degenerate primer pools are used or when mismatches between primers and template are possible. PMID:25996930
-
Origin and quantification of circulating DNA in mice with human colorectal cancer xenografts
Thierry, Alain R.; Mouliere, Florent; Gongora, Celine; Ollier, Jeremy; Robert, Bruno; Ychou, Marc; Del Rio, Maguy; Molina, Franck
2010-01-01
Although circulating DNA (ctDNA) could be an attractive tool for early cancer detection, diagnosis, prognosis, monitoring or prediction of response to therapies, knowledge on its origin, form and rate of release is poor and often contradictory. Here, we describe an experimental system to systematically examine these aspects. Nude mice were xenografted with human HT29 or SW620 colorectal carcinoma (CRC) cells and ctDNA was analyzed by Q–PCR with highly specific and sensitive primer sets at different times post-graft. We could discriminate ctDNA from normal (murine) cells and from mutated and non-mutated tumor (human) cells by using species-specific KRAS or PSAT1 primers and by assessing the presence of the BRAF V600E mutation. The concentration of human (mutated and non-mutated) ctDNA increased significantly with tumor growth. Conversely, and differently from previous studies, low, constant level of mouse ctDNA was observed, thus facilitating the study of mutated and non-mutated tumor derived ctDNA. Finally, analysis of ctDNA fragmentation confirmed the predominance of low-size fragments among tumor ctDNA from mice with bigger tumors. Higher ctDNA fragmentation was also observed in plasma samples from three metastatic CRC patients in comparison to healthy individuals. Our data confirm the predominance of mononucleosome-derived fragments in plasma from xenografted animals and, as a consequence, of apoptosis as a source of ctDNA, in particular for tumor-derived ctDNA. Altogether, our results suggest that ctDNA features vary during CRC tumor development and our experimental system might be a useful tool to follow such variations. PMID:20494973
-
Sanjay, Archana; Miyazaki, Tsuyoshi; Itzstein, Cecile; Purev, Enkhtsetseg; Horne, William C; Baron, Roland
2006-12-01
Cbl is an adaptor protein and ubiquitin ligase that binds and is phosphorylated by the nonreceptor tyrosine kinase Src. We previously showed that the primary interaction between Src and Cbl is mediated by the Src homology domain 3 (SH3) of Src binding to proline-rich sequences of Cbl. The peptide Cbl RDLPPPPPPDRP(540-551), which corresponds to residues 540-551 of Cbl, inhibited the binding of a GST-Src SH3 fusion protein to Cbl, whereas RDLAPPAPPPDR(540-551) did not, suggesting that Src binds to this site on Cbl in a class I orientation. Mutating prolines 543-548 reduced Src binding to the Cbl 479-636 fragment significantly more than mutating the prolines in the PPVPPR(494-499) motif, which was previously reported to bind Src SH3. Mutating Cbl prolines 543-548 to alanines substantially reduced Src binding to Cbl, Src-induced phosphorylation of Cbl, and the inhibition of Src kinase activity by Cbl. Expressing the mutated Cbl in osteoclasts induced a moderate reduction in bone-resorbing activity and increased amounts of Src protein. In contrast, disabling the tyrosine kinase-binding domain of full-length Cbl by mutating glycine 306 to glutamic acid, and thereby preventing the previously described binding of the tyrosine kinase-binding domain to the Src phosphotyrosine 416, had no effect on Cbl phosphorylation, the inhibition of Src activity by full-length Cbl, or bone resorption. These data indicate that the Cbl RDLPPPP(540-546) sequence is a functionally important binding site for Src.
-
A rapid screening with direct sequencing from blood samples for the diagnosis of Leigh syndrome.
Shimbo, Hiroko; Takagi, Mariko; Okuda, Mitsuko; Tsuyusaki, Yu; Takano, Kyoko; Iai, Mizue; Yamashita, Sumimasa; Murayama, Kei; Ohtake, Akira; Goto, Yu-Ichi; Aida, Noriko; Osaka, Hitoshi
2014-01-01
Large numbers of genes are responsible for Leigh syndrome (LS), making genetic confirmation of LS difficult. We screened our patients with LS using a limited set of 21 primers encompassing the frequently reported gene for the respiratory chain complexes I (ND1-ND6, and ND4L), IV(SURF1), and V(ATP6) and the pyruvate dehydrogenase E1α-subunit. Of 18 LS patients, we identified mutations in 11 patients, including 7 in mDNA (two with ATP6), 4 in nuclear (three with SURF1). Overall, we identified mutations in 61% of LS patients (11/18 individuals) in this cohort. Sanger sequencing with our limited set of primers allowed us a rapid genetic confirmation of more than half of the LS patients and it appears to be efficient as a primary genetic screening in this cohort.
-
Molecular basis of splotch and Waardenburg Pax-3 mutations.
Chalepakis, G; Goulding, M; Read, A; Strachan, T; Gruss, P
1994-01-01
Pax genes control certain aspects of development, as mutations result in (semi)dominant defects apparent during embryogenesis. Pax-3 has been associated with the mouse mutant splotch (Sp) and the human Waardenburg syndrome type 1 (WS1). We have examined the molecular basis of splotch and WS1 by studying the effect of mutations on DNA binding, using a defined target sequence. Pax-3 contains two different types of functional DNA-binding domains, a paired domain and a homeodomain. Mutational analysis of Pax-3 reveals different modes of DNA binding depending on the presence of these domains. A segment of Pax-3 located between the two DNA-binding domains, including a conserved octapeptide, participates in protein homodimerization. Pax-3 mutations found in splotch alleles and WS1 individuals change DNA binding and, in the case of a protein product of the Sp allele, dimerization. These findings were taken as a basis to define the molecular nature of the mutants. Images PMID:7909605
-
Varghese, Leila N; Zhang, Jian-Guo; Young, Samuel N; Willson, Tracy A; Alexander, Warren S; Nicola, Nicos A; Babon, Jeffrey J; Murphy, James M
2014-02-01
Activation of the cell surface receptor, c-Mpl, by the cytokine, thrombopoietin (TPO), underpins megakaryocyte and platelet production in mammals. In humans, mutations in c-Mpl have been identified as the molecular basis of Congenital Amegakaryocytic Thrombocytopenia (CAMT). Here, we show that CAMT-associated mutations in c-Mpl principally lead to defective receptor presentation on the cell surface. In contrast, one CAMT mutant c-Mpl, F104S, was expressed on the cell surface, but showed defective TPO binding and receptor activation. Using mutational analyses, we examined which residues adjacent to F104 within the membrane-distal cytokine receptor homology module (CRM) of c-Mpl comprise the TPO-binding epitope, revealing residues within the predicted Domain 1 E-F and A-B loops and Domain 2 F'-G' loop as key TPO-binding determinants. These studies underscore the importance of the c-Mpl membrane-distal CRM to TPO-binding and suggest that mutations within this CRM that perturb TPO binding could give rise to CAMT.
-
Du, Yushen; Zhang, Tian-Hao; Dai, Lei; Zheng, Xiaojuan; Gorin, Aleksandr M; Oishi, John; Wu, Ting-Ting; Yoshizawa, Janice M; Li, Xinmin; Yang, Otto O; Martinez-Maza, Otoniel; Detels, Roger; Sun, Ren
2017-11-28
Certain "protective" major histocompatibility complex class I (MHC-I) alleles, such as B*57 and B*27, are associated with long-term control of HIV-1 in vivo mediated by the CD8 + cytotoxic-T-lymphocyte (CTL) response. However, the mechanism of such superior protection is not fully understood. Here we combined high-throughput fitness profiling of mutations in HIV-1 Gag, in silico prediction of MHC-peptide binding affinity, and analysis of intraperson virus evolution to systematically compare differences with respect to CTL escape mutations between epitopes targeted by protective MHC-I alleles and those targeted by nonprotective MHC-I alleles. We observed that the effects of mutations on both viral replication and MHC-I binding affinity are among the determinants of CTL escape. Mutations in Gag epitopes presented by protective MHC-I alleles are associated with significantly higher fitness cost and lower reductions in binding affinity with respect to MHC-I. A linear regression model accounting for the effect of mutations on both viral replicative capacity and MHC-I binding can explain the protective efficacy of MHC-I alleles. Finally, we found a consistent pattern in the evolution of Gag epitopes in long-term nonprogressors versus progressors. Overall, our results suggest that certain protective MHC-I alleles allow superior control of HIV-1 by targeting epitopes where mutations typically incur high fitness costs and small reductions in MHC-I binding affinity. IMPORTANCE Understanding the mechanism of viral control achieved in long-term nonprogressors with protective HLA alleles provides insights for developing functional cure of HIV infection. Through the characterization of CTL escape mutations in infected persons, previous researchers hypothesized that protective alleles target epitopes where escape mutations significantly reduce viral replicative capacity. However, these studies were usually limited to a few mutations observed in vivo Here we utilized our recently developed high-throughput fitness profiling method to quantitatively measure the fitness of mutations across the entirety of HIV-1 Gag. The data enabled us to integrate the results with in silico prediction of MHC-peptide binding affinity and analysis of intraperson virus evolution to systematically determine the differences in CTL escape mutations between epitopes targeted by protective HLA alleles and those targeted by nonprotective HLA alleles. We observed that the effects of Gag epitope mutations on HIV replicative fitness and MHC-I binding affinity are among the major determinants of CTL escape. Copyright © 2017 Du et al.
-
Long, Katherine S; Poehlsgaard, Jacob; Hansen, Lykke H; Hobbie, Sven N; Böttger, Erik C; Vester, Birte
2009-03-01
Tiamulin and valnemulin target the peptidyl transferase centre (PTC) on the bacterial ribosome. They are used in veterinary medicine to treat infections caused by a variety of bacterial pathogens, including the intestinal spirochetes Brachyspira spp. Mutations in ribosomal protein L3 and 23S rRNA have previously been associated with tiamulin resistance in Brachyspira spp. isolates, but as multiple mutations were isolated together, the roles of the individual mutations are unclear. In this work, individual 23S rRNA mutations associated with pleuromutilin resistance at positions 2055, 2447, 2504 and 2572 (Escherichia coli numbering) are introduced into a Mycobacterium smegmatis strain with a single rRNA operon. The single mutations each confer a significant and similar degree of valnemulin resistance and those at 2447 and 2504 also confer cross-resistance to other antibiotics that bind to the PTC in M. smegmatis. Antibiotic footprinting experiments on mutant ribosomes show that the introduced mutations cause structural perturbations at the PTC and reduced binding of pleuromutilin antibiotics. This work underscores the fact that mutations at nucleotides distant from the pleuromutilin binding site can confer the same level of valnemulin resistance as those at nucleotides abutting the bound drug, and suggests that the former function indirectly by altering local structure and flexibility at the drug binding pocket.
-
Gao, Yong-Gui; Suzuki, Hiroaki; Itou, Hiroshi; Zhou, Yong; Tanaka, Yoshikazu; Wachi, Masaaki; Watanabe, Nobuhisa; Tanaka, Isao; Yao, Min
2008-01-01
LldR (CGL2915) from Corynebacterium glutamicum is a transcription factor belonging to the GntR family, which is typically involved in the regulation of oxidized substrates associated with amino acid metabolism. In the present study, the crystal structure of LldR was determined at 2.05-Å resolution. The structure consists of N- and C-domains similar to those of FadR, but with distinct domain orientations. LldR and FadR dimers achieve similar structures by domain swapping, which was first observed in dimeric assembly of transcription factors. A structural feature of Zn2+ binding in the regulatory domain was also observed, as a difference from the FadR subfamily. DNA microarray and DNase I footprint analyses suggested that LldR acts as a repressor regulating cgl2917-lldD and cgl1934-fruK-ptsF operons, which are indispensable for l-lactate and fructose/sucrose utilization, respectively. Furthermore, the stoichiometries and affinities of LldR and DNAs were determined by isothermal titration calorimetry measurements. The transcriptional start site and repression of LldR on the cgl2917-lldD operon were analysed by primer extension assay. Mutation experiments showed that residues Lys4, Arg32, Arg42 and Gly63 are crucial for DNA binding. The location of the putative ligand binding cavity and the regulatory mechanism of LldR on its affinity for DNA were proposed. PMID:18988622
-
Sensitive and reliable detection of Kit point mutation Asp 816 to Val in pathological material
Kähler, Christian; Didlaukat, Sabine; Feller, Alfred C; Merz, Hartmut
2007-01-01
Background Human mastocytosis is a heterogenous disorder which is linked to a gain-of-function mutation in the kinase domain of the receptor tyrosine kinase Kit. This D816V mutation leads to constitutive activation and phosphorylation of Kit with proliferative disorders of mast cells in the peripheral blood, skin, and spleen. Most PCR applications used so far are labour-intensive and are not adopted to daily routine in pathological laboratories. The method has to be robust and working on such different materials like archival formalin-fixed, paraffin-embedded tissue (FFPE) and blood samples. Such a method is introduced in this publication. Methods The Kit point mutation Asp 816 to Val is heterozygous which means a problem in detection by PCR because the wild-type allele is also amplified and the number of cells which bear the point mutation is in most of the cases low. Most PCR protocols use probes to block the wild-type allele during amplification with more or less satisfying result. This is why point-mutated forward primers were designed and tested for efficiency in amplification of the mutated allele. Results One primer combination (A) fits the most for the introduced PCR assay. It was able just to amplify the mutated allele with high specificity from different patient's materials (FFPE or blood) of varying quality and quantity. Moreover, the sensitivity for this assay was convincing because 10 ng of DNA which bears the point mutation could be detected in a total volume of 200 ng of DNA. Conclusion The PCR assay is able to deal with different materials (blood and FFPE) this means quality and quantity of DNA and can be used for high-througput screening because of its robustness. Moreover, the method is easy-to-use, not labour-intensive, and easy to realise in a standard laboratory. PMID:17900365
-
Kennedy, Melissa A; Moore, Emily; Wilkes, Rebecca P; Citino, Scott B; Kania, Stephen A
2006-04-01
To analyze the 7a7b genes of the feline coronavirus (FCoV) of cheetahs, which are believed to play a role in virulence of this virus. Biologic samples collected during a 4-year period from 5 cheetahs at the same institution and at 1 time point from 4 cheetahs at different institutions. Samples were first screened for FCoV via a reverse transcription-PCR procedure involving primers that encompassed the 3'-untranslated region. Samples that yielded positive assay results were analyzed by use of primers that targeted the 7a7b open reading frames. The nucleotide sequences of the 7a7b amplification products were determined and analyzed. In most isolates, substantial deletional mutations in the 7a gene were detected that would result in aberrant or no expression of the 7a product because of altered reading frames. Although the 7b gene was also found to contain mutations, these were primarily point mutations resulting in minor amino acid changes. The coronavirus associated with 1 cheetah with feline infectious peritonitis had intact 7a and 7b genes. The data suggest that mutations arise readily in the 7a region and may remain stable in FCoV of cheetahs. In contrast, an intact 7b gene may be necessary for in vivo virus infection and replication. Persistent infection with FCoV in a cheetah population results in continued virus circulation and may lead to a quasispecies of virus variants.
-
Role of the C-terminal residue of the DNA polymerase of bacteriophage T7.
Kumar, J K; Tabor, S; Richardson, C C
2001-09-14
The crystal structure of the DNA polymerase encoded by gene 5 of bacteriophage T7, in a complex with its processivity factor, Escherichia coli thioredoxin, a primer-template, and an incoming deoxynucleoside triphosphate reveals a putative hydrogen bond between the C-terminal residue, histidine 704 of gene 5 protein, and an oxygen atom on the penultimate phosphate diester of the primer strand. Elimination of this electrostatic interaction by replacing His(704) with alanine renders the phage nonviable, and no DNA synthesis is observed in vivo. Polymerase activity of the genetically altered enzyme on primed M13 DNA is only 12% of the wild-type enzyme, and its processivity is drastically reduced. Kinetic parameters for binding a primer-template (K(D)(app)), nucleotide binding (K(m)), and k(off) for dissociation of the altered polymerase from a primer-template are not significantly different from that of wild-type T7 DNA polymerase. However, the decrease in polymerase activity is concomitant with increased hydrolytic activity, judging from the turnover of nucleoside triphosphate into the corresponding nucleoside monophosphate (percentage of turnover, 65%) during DNA synthesis. Biochemical data along with structural observations imply that the terminal amino acid residue of T7 DNA polymerase plays a critical role in partitioning DNA between the polymerase and exonuclease sites.
-
Er, Tze-Kiong; Chen, Chih-Chieh; Liu, Yen-Yi; Chang, Hui-Chiu; Chien, Yin-Hsiu; Chang, Jan-Gowth; Hwang, Jenn-Kang; Jong, Yuh-Jyh
2011-10-21
Multiple acyl-coenzyme A dehydrogenase deficiency (MADD) is an autosomal recessive disease caused by the defects in the mitochondrial electron transfer system and the metabolism of fatty acids. Recently, mutations in electron transfer flavoprotein dehydrogenase (ETFDH) gene, encoding electron transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO) have been reported to be the major causes of riboflavin-responsive MADD. To date, no studies have been performed to explore the functional impact of these mutations or their mechanism of disrupting enzyme activity. High resolution melting (HRM) analysis and sequencing of the entire ETFDH gene revealed a novel mutation (p.Phe128Ser) and the hotspot mutation (p.Ala84Thr) from a patient with MADD. According to the predicted 3D structure of ETF:QO, the two mutations are located within the flavin adenine dinucleotide (FAD) binding domain; however, the two residues do not have direct interactions with the FAD ligand. Using molecular dynamics (MD) simulations and normal mode analysis (NMA), we found that the p.Ala84Thr and p.Phe128Ser mutations are most likely to alter the protein structure near the FAD binding site as well as disrupt the stability of the FAD binding required for the activation of ETF:QO. Intriguingly, NMA revealed that several reported disease-causing mutations in the ETF:QO protein show highly correlated motions with the FAD-binding site. Based on the present findings, we conclude that the changes made to the amino acids in ETF:QO are likely to influence the FAD-binding stability.
-
2011-01-01
Background Multiple acyl-coenzyme A dehydrogenase deficiency (MADD) is an autosomal recessive disease caused by the defects in the mitochondrial electron transfer system and the metabolism of fatty acids. Recently, mutations in electron transfer flavoprotein dehydrogenase (ETFDH) gene, encoding electron transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO) have been reported to be the major causes of riboflavin-responsive MADD. To date, no studies have been performed to explore the functional impact of these mutations or their mechanism of disrupting enzyme activity. Results High resolution melting (HRM) analysis and sequencing of the entire ETFDH gene revealed a novel mutation (p.Phe128Ser) and the hotspot mutation (p.Ala84Thr) from a patient with MADD. According to the predicted 3D structure of ETF:QO, the two mutations are located within the flavin adenine dinucleotide (FAD) binding domain; however, the two residues do not have direct interactions with the FAD ligand. Using molecular dynamics (MD) simulations and normal mode analysis (NMA), we found that the p.Ala84Thr and p.Phe128Ser mutations are most likely to alter the protein structure near the FAD binding site as well as disrupt the stability of the FAD binding required for the activation of ETF:QO. Intriguingly, NMA revealed that several reported disease-causing mutations in the ETF:QO protein show highly correlated motions with the FAD-binding site. Conclusions Based on the present findings, we conclude that the changes made to the amino acids in ETF:QO are likely to influence the FAD-binding stability. PMID:22013910
-
DOE Office of Scientific and Technical Information (OSTI.GOV)
Borucki, Monica K.; Lao, Victoria; Hwang, Mona
Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging human pathogen related to SARS virus. In vitro studies indicate this virus may have a broad host range suggesting an increased pandemic potential. Genetic and epidemiological evidence indicate camels serve as a reservoir for MERS virus but the mechanism of cross species transmission is unclear and many questions remain regarding the susceptibility of humans to infection. Deep sequencing data was obtained from the nasal samples of three camels that had been experimentally infected with a human MERS-CoV isolate. A majority of the genome was covered and average coverage was greater thanmore » 12,000x depth. Although only 5 mutations were detected in the consensus sequences, 473 intrahost single nucleotide variants were identified. Lastly, many of these variants were present at high frequencies and could potentially influence viral phenotype and the sensitivity of detection assays that target these regions for primer or probe binding.« less
-
Schein, Catherine H; Ye, Mengyi; Paul, Aniko V; Oberste, M Steven; Chapman, Nora; van der Heden van Noort, Gerbrand J; Filippov, Dmitri V; Choi, Kyung H
2015-10-01
Enteroviruses (EV) uridylylate a peptide, VPg, as the first step in their replication. VPgpUpU, found free in infected cells, serves as the primer for RNA elongation. The abilities of four polymerases (3D(pol)), from EV-species A-C, to uridylylate VPgs that varied by up to 60% of their residues were compared. Each 3D(pol) was able to uridylylate all five VPgs using polyA RNA as template, while showing specificity for its own genome encoded peptide. All 3D(pol) uridylylated a consensus VPg representing the physical chemical properties of 31 different VPgs. Thus the residues required for uridylylation and the enzymatic mechanism must be similar in diverse EV. As VPg-binding sites differ in co-crystal structures, the reaction is probably done by a second 3D(pol) molecule. The conservation of polymerase residues whose mutation reduces uridylylation but not RNA elongation is compared. Copyright © 2015 Elsevier Inc. All rights reserved.
-
Borucki, Monica K.; Lao, Victoria; Hwang, Mona; ...
2016-01-20
Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging human pathogen related to SARS virus. In vitro studies indicate this virus may have a broad host range suggesting an increased pandemic potential. Genetic and epidemiological evidence indicate camels serve as a reservoir for MERS virus but the mechanism of cross species transmission is unclear and many questions remain regarding the susceptibility of humans to infection. Deep sequencing data was obtained from the nasal samples of three camels that had been experimentally infected with a human MERS-CoV isolate. A majority of the genome was covered and average coverage was greater thanmore » 12,000x depth. Although only 5 mutations were detected in the consensus sequences, 473 intrahost single nucleotide variants were identified. Lastly, many of these variants were present at high frequencies and could potentially influence viral phenotype and the sensitivity of detection assays that target these regions for primer or probe binding.« less
-
Loussouarn, Delphine; Le Loupp, Anne-Gaëlle; Frenel, Jean-Sébastien; Leclair, François; Von Deimling, Andreas; Aumont, Maud; Martin, Stéphane; Campone, Mario; Denis, Marc G
2012-06-01
Previous studies have identified mutations of the isocitrate dehydrogenase 1 (IDH1) gene in more than 70% of World Health Organization (WHO) grade II and III gliomas. The most frequent mutation leads to a specific amino acid change from arginine to histidine at codon 132 (c.395G>A, p.R132H). IDH1 mutated tumors have a better prognosis than IDH1 non-mutated tumors. The aim of our study was to evaluate and compare the methods of mIDH1 R132H immunohistochemistry, allele-specific PCR and DNA sequencing for determination of IDH1 status. We performed a retrospective study of 91 patients with WHO grade II (n=43) and III (n=48) oligodendrogliomas. A fragment of exon 4 spanning the sequence encoding the catalytic domain of IDH1, including codon 132, was amplified and sequenced using standard conditions. Allele-specific amplification was performed using two forward primers with variations in their 3' nucleotides such that each was specific for the wild-type or the mutated variant, and one reverse primer. Immunohistochemistry was performed with mouse monoclonal mIDH1 R132H. DNA was extracted from FFPE sections following macrodissection. IDH1 mutations were found in 55/90 patients (61.1%) by direct sequencing. R132H mutations were found in 47/55 patients (85.4%). The results of the allele-specific PCR positively correlated with those from DNA sequencing. Other mutations (p.R132C, p.R132S and pR132G) were found by DNA sequencing in 3, 3 and 2 tumors, respectively (8/55 patients, 14.6%). mIDH1 R132H immunostaining was found in the 47 patients presenting the R132H mutation (sensitivity 47/47, 100% for this mutation). None of the tumors presenting a wild-type IDH1 gene were stained (specificity 35/35, 100%). Our results demonstrate that immunohistochemistry using the mIDH1 R132H antibody and allele-specific amplification are highly sensitive techniques to detect the most frequent mutation of the IDH1 gene.
-
Structure of adenovirus bound to cellular receptor car
Freimuth, Paul I.
2004-05-18
Disclosed is a mutant adenovirus which has a genome comprising one or more mutations in sequences which encode the fiber protein knob domain wherein the mutation causes the encoded viral particle to have significantly weakened binding affinity for CARD1 relative to wild-type adenovirus. Such mutations may be in sequences which encode either the AB loop, or the HI loop of the fiber protein knob domain. Specific residues and mutations are described. Also disclosed is a method for generating a mutant adenovirus which is characterized by a receptor binding affinity or specificity which differs substantially from wild type. In the method, residues of the adenovirus fiber protein knob domain which are predicted to alter D1 binding when mutated, are identified from the crystal structure coordinates of the AD12knob:CAR-D1 complex. A mutation which alters one or more of the identified residues is introduced into the genome of the adenovirus to generate a mutant adenovirus. Whether or not the mutant produced exhibits altered adenovirus-CAR binding properties is then determined.
-
Koenig, Patrick; Lee, Chingwei V.; Walters, Benjamin T.; Janakiraman, Vasantharajan; Stinson, Jeremy; Patapoff, Thomas W.; Fuh, Germaine
2017-01-01
Somatic mutations within the antibody variable domains are critical to the immense capacity of the immune repertoire. Here, via a deep mutational scan, we dissect how mutations at all positions of the variable domains of a high-affinity anti-VEGF antibody G6.31 impact its antigen-binding function. The resulting mutational landscape demonstrates that large portions of antibody variable domain positions are open to mutation, and that beneficial mutations can be found throughout the variable domains. We determine the role of one antigen-distal light chain position 83, demonstrating that mutation at this site optimizes both antigen affinity and thermostability by modulating the interdomain conformational dynamics of the antigen-binding fragment. Furthermore, by analyzing a large number of human antibody sequences and structures, we demonstrate that somatic mutations occur frequently at position 83, with corresponding domain conformations observed for G6.31. Therefore, the modulation of interdomain dynamics represents an important mechanism during antibody maturation in vivo. PMID:28057863
-
McCutchen-Maloney, Sandra L.
2002-01-01
DNA mutation binding proteins alone and as chimeric proteins with nucleases are used with solid supports to detect DNA sequence variations, DNA mutations and single nucleotide polymorphisms. The solid supports may be flow cytometry beads, DNA chips, glass slides or DNA dips sticks. DNA molecules are coupled to solid supports to form DNA-support complexes. Labeled DNA is used with unlabeled DNA mutation binding proteins such at TthMutS to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by binding which gives an increase in signal. Unlabeled DNA is utilized with labeled chimeras to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by nuclease activity of the chimera which gives a decrease in signal.
-
Prokop, Susanne; Perry, Nicole A; Vishnivetskiy, Sergey A; Toth, Andras D; Inoue, Asuka; Milligan, Graeme; Iverson, Tina M; Hunyady, Laszlo; Gurevich, Vsevolod V
2017-08-01
Non-visual arrestins interact with hundreds of different G protein-coupled receptors (GPCRs). Here we show that by introducing mutations into elements that directly bind receptors, the specificity of arrestin-3 can be altered. Several mutations in the two parts of the central "crest" of the arrestin molecule, middle-loop and C-loop, enhanced or reduced arrestin-3 interactions with several GPCRs in receptor subtype and functional state-specific manner. For example, the Lys139Ile substitution in the middle-loop dramatically enhanced the binding to inactive M 2 muscarinic receptor, so that agonist activation of the M 2 did not further increase arrestin-3 binding. Thus, the Lys139Ile mutation made arrestin-3 essentially an activation-independent binding partner of M 2 , whereas its interactions with other receptors, including the β 2 -adrenergic receptor and the D 1 and D 2 dopamine receptors, retained normal activation dependence. In contrast, the Ala248Val mutation enhanced agonist-induced arrestin-3 binding to the β 2 -adrenergic and D 2 dopamine receptors, while reducing its interaction with the D 1 dopamine receptor. These mutations represent the first example of altering arrestin specificity via enhancement of the arrestin-receptor interactions rather than selective reduction of the binding to certain subtypes. Copyright © 2017. Published by Elsevier Inc.
-
Sawamura, Kensuke; Hashimoto, Masahiko
2017-01-01
A fluorescence quenching assay based on a ligase detection reaction was developed for facile and rapid detection of point mutations present in a mixed population of non-variant DNA. If the test DNA carried a targeted mutation, then the two allele-specific primers were ligated to form a molecular beacon resulting in the expected fluorescence quenching signatures. Using this method, we successfully detected as low as 5% mutant DNA in a mixture of wild-type DNA (t test at 99% confidence level).
-
Structure of the coding region and mRNA variants of the apyrase gene from pea (Pisum sativum)
NASA Technical Reports Server (NTRS)
Shibata, K.; Abe, S.; Davies, E.
2001-01-01
Partial amino acid sequences of a 49 kDa apyrase (ATP diphosphohydrolase, EC 3.6.1.5) from the cytoskeletal fraction of etiolated pea stems were used to derive oligonucleotide DNA primers to generate a cDNA fragment of pea apyrase mRNA by RT-PCR and these primers were used to screen a pea stem cDNA library. Two almost identical cDNAs differing in just 6 nucleotides within the coding regions were found, and these cDNA sequences were used to clone genomic fragments by PCR. Two nearly identical gene fragments containing 8 exons and 7 introns were obtained. One of them (H-type) encoded the mRNA sequence described by Hsieh et al. (1996) (DDBJ/EMBL/GenBank Z32743), while the other (S-type) differed by the same 6 nucleotides as the mRNAs, suggesting that these genes may be alleles. The six nucleotide differences between these two alleles were found solely in the first exon, and these mutation sites had two types of consensus sequences. These mRNAs were found with varying lengths of 3' untranslated regions (3'-UTR). There are some similarities between the 3'-UTR of these mRNAs and those of actin and actin binding proteins in plants. The putative roles of the 3'-UTR and alternative polyadenylation sites are discussed in relation to their possible role in targeting the mRNAs to different subcellular compartments.
-
Luo, Xiao-Feng; Jiao, Jian-Hua; Zhang, Wen-Yue; Pu, Han-Ming; Qu, Bao-Jin; Yang, Bing-Ya; Hou, Min; Ji, Min-Jun
2016-07-07
To investigate clarithromycin resistance positions 2142, 2143 and 2144 of the 23SrRNA gene in Helicobacter pylori (H. pylori) by nested-allele specific primer-polymerase chain reaction (nested-ASP-PCR). The gastric tissue and saliva samples from 99 patients with positive results of the rapid urease test (RUT) were collected. The nested-ASP-PCR method was carried out with the external primers and inner allele-specific primers corresponding to the reference strain and clinical strains. Thirty gastric tissue and saliva samples were tested to determine the sensitivity of nested-ASP-PCR and ASP-PCR methods. Then, clarithromycin resistance was detected for 99 clinical samples by using different methods, including nested-ASP-PCR, bacterial culture and disk diffusion. The nested-ASP-PCR method was successfully established to test the resistance mutation points 2142, 2143 and 2144 of the 23SrRNA gene of H. pylori. Among 30 samples of gastric tissue and saliva, the H. pylori detection rate of nested-ASP-PCR was 90% and 83.33%, while the detection rate of ASP-PCR was just 63% and 56.67%. Especially in the saliva samples, nested-ASP-PCR showed much higher sensitivity in H. pylori detection and resistance mutation rates than ASP-PCR. In the 99 RUT-positive gastric tissue and saliva samples, the H. pylori-positive detection rate by nested-ASP-PCR was 87 (87.88%) and 67 (67.68%), in which there were 30 wild-type and 57 mutated strains in gastric tissue and 22 wild-type and 45 mutated strains in saliva. Genotype analysis showed that three-points mixed mutations were quite common, but different resistant strains were present in gastric mucosa and saliva. Compared to the high sensitivity shown by nested-ASP-PCR, the positive detection of bacterial culture with gastric tissue samples was 50 cases, in which only 26 drug-resistant strains were found through analyzing minimum inhibitory zone of clarithromycin. The nested-ASP-PCR assay showed higher detection sensitivity than ASP-PCR and drug sensitivity testing, which could be performed to evaluate clarithromycin resistance of H. pylori.
-
Luo, Xiao-Feng; Jiao, Jian-Hua; Zhang, Wen-Yue; Pu, Han-Ming; Qu, Bao-Jin; Yang, Bing-Ya; Hou, Min; Ji, Min-Jun
2016-01-01
AIM: To investigate clarithromycin resistance positions 2142, 2143 and 2144 of the 23SrRNA gene in Helicobacter pylori (H. pylori) by nested-allele specific primer-polymerase chain reaction (nested-ASP-PCR). METHODS: The gastric tissue and saliva samples from 99 patients with positive results of the rapid urease test (RUT) were collected. The nested-ASP-PCR method was carried out with the external primers and inner allele-specific primers corresponding to the reference strain and clinical strains. Thirty gastric tissue and saliva samples were tested to determine the sensitivity of nested-ASP-PCR and ASP-PCR methods. Then, clarithromycin resistance was detected for 99 clinical samples by using different methods, including nested-ASP-PCR, bacterial culture and disk diffusion. RESULTS: The nested-ASP-PCR method was successfully established to test the resistance mutation points 2142, 2143 and 2144 of the 23SrRNA gene of H. pylori. Among 30 samples of gastric tissue and saliva, the H. pylori detection rate of nested-ASP-PCR was 90% and 83.33%, while the detection rate of ASP-PCR was just 63% and 56.67%. Especially in the saliva samples, nested-ASP-PCR showed much higher sensitivity in H. pylori detection and resistance mutation rates than ASP-PCR. In the 99 RUT-positive gastric tissue and saliva samples, the H. pylori-positive detection rate by nested-ASP-PCR was 87 (87.88%) and 67 (67.68%), in which there were 30 wild-type and 57 mutated strains in gastric tissue and 22 wild-type and 45 mutated strains in saliva. Genotype analysis showed that three-points mixed mutations were quite common, but different resistant strains were present in gastric mucosa and saliva. Compared to the high sensitivity shown by nested-ASP-PCR, the positive detection of bacterial culture with gastric tissue samples was 50 cases, in which only 26 drug-resistant strains were found through analyzing minimum inhibitory zone of clarithromycin. CONCLUSION: The nested-ASP-PCR assay showed higher detection sensitivity than ASP-PCR and drug sensitivity testing, which could be performed to evaluate clarithromycin resistance of H. pylori. PMID:27433095
-
Garcia, J A; Harrich, D; Soultanakis, E; Wu, F; Mitsuyasu, R; Gaynor, R B
1989-01-01
The human immunodeficiency virus (HIV) type 1 LTR is regulated at the transcriptional level by both cellular and viral proteins. Using HeLa cell extracts, multiple regions of the HIV LTR were found to serve as binding sites for cellular proteins. An untranslated region binding protein UBP-1 has been purified and fractions containing this protein bind to both the TAR and TATA regions. To investigate the role of cellular proteins binding to both the TATA and TAR regions and their potential interaction with other HIV DNA binding proteins, oligonucleotide-directed mutagenesis of both these regions was performed followed by DNase I footprinting and transient expression assays. In the TATA region, two direct repeats TC/AAGC/AT/AGCTGC surround the TATA sequence. Mutagenesis of both of these direct repeats or of the TATA sequence interrupted binding over the TATA region on the coding strand, but only a mutation of the TATA sequence affected in vivo assays for tat-activation. In addition to TAR serving as the site of binding of cellular proteins, RNA transcribed from TAR is capable of forming a stable stem-loop structure. To determine the relative importance of DNA binding proteins as compared to secondary structure, oligonucleotide-directed mutations in the TAR region were studied. Local mutations that disrupted either the stem or loop structure were defective in gene expression. However, compensatory mutations which restored base pairing in the stem resulted in complete tat-activation. This indicated a significant role for the stem-loop structure in HIV gene expression. To determine the role of TAR binding proteins, mutations were constructed which extensively changed the primary structure of the TAR region, yet left stem base pairing, stem energy and the loop sequence intact. These mutations resulted in decreased protein binding to TAR DNA and defects in tat-activation, and revealed factor binding specifically to the loop DNA sequence. Further mutagenesis which inverted this stem and loop mutation relative to the HIV LTR mRNA start site resulted in even larger decreases in tat-activation. This suggests that multiple determinants, including protein binding, the loop sequence, and RNA or DNA secondary structure, are important in tat-activation and suggests that tat may interact with cellular proteins binding to DNA to increase HIV gene expression. Images PMID:2721501
-
Structure of adenovirus bound to cellular receptor car
Freimuth, Paul I.
2007-01-02
Disclosed is a mutant CAR-DI-binding adenovirus which has a genome comprising one or more mutations in sequences which encode the fiber protein knob domain wherein the mutation causes the encoded viral particle to have a significantly weakened binding affinity for CAR-DI relative to wild-type adenovirus. Such mutations may be in sequences which encode either the AB loop, or the HI loop of the fiber protein knob domain. Specific residues and mutations are described. Also disclosed is a method for generating a mutant adenovirus which is characterized by a receptor binding affinity or specificity which differs substantially from wild type.
-
Chandra, Saurabh; Kapur, Reuben; Chuzhanova, Nadia; Summey, Victoria; Prentice, David; Barker, Jane; Cooper, David N; Williams, David A
2003-11-15
Kit ligand (Kitl), encoded by the Steel (Sl) locus, plays an essential role in hematopoiesis, gametogenesis, and melanogenesis during both embryonic and adult life. We have characterized a new spontaneous mutant of the Sl locus in mice designated KitlSl-20J that arose in the breeding colony at Jackson Laboratories. Heterozygous KitlSl-20J mice display a white belly spot and intercrossing results in an embryonic lethal phenotype in the homozygous state. Analysis of homozygous embryos demonstrated a significant reduction in fetal liver cellularity, colony forming unit-erythroid (CFU-E) progenitors, and a total absence of germ cells. Although expressed in vivo, recombinant mutant protein demonstrated loss of bioactivity that was correlated with lack of receptor binding. Analysis of the Sl gene transcripts in heterozygous KitlSl-20J mice revealed an in-frame tandem duplication of exon 3. A long-range polymerase chain reaction (PCR) strategy using overlapping primers in exon 3 amplified an approximately 7-kilobase (kb) product from DNA isolated from heterozygous KitlSl-20J mice but not from wild-type DNA that contained sequences from both introns 2 and 3 and an inverted intron 2 sequence, suggesting a complex rearrangement as the mechanism of the mutation. "Complexity analysis" of the sequence of the amplified product strongly suggests that local DNA motifs may have contributed to the generation of this spontaneous KitlSl-20J allele, likely mediated by a 2-step process. The KitlSl-20J mutation is a unique KitlSl allele and represents an unusual mechanism of mutation.
-
DOE Office of Scientific and Technical Information (OSTI.GOV)
Sine, Steven M.; Huang, Sun; Li, Shu-Xing
2013-09-01
The crystal structure of a pentameric α7 ligand-binding domain chimaera with bound α-btx (α-bungarotoxin) showed that of the five conserved aromatic residues in α7, only Tyr 184 in loop C of the ligand-binding site was required for high-affinity binding. To determine whether the contribution of Tyr 184 depends on local residues, we generated mutations in an α7/5HT 3A (5-hydroxytryptamine type 3A) receptor chimaera, individually and in pairs, and measured 125I-labelled α-btx binding. The results show that mutations of individual residues near Tyr 184 do not affect α-btx affinity, but pairwise mutations decrease affinity in an energetically coupled manner. Kinetic measurementsmore » show that the affinity decreases arise through increases in the α-btx dissociation rate with little change in the association rate. Replacing loop C in α7 with loop C from the α-btx-insensitive α2 or α3 subunits abolishes high-affinity α-btx binding, but preserves acetylcholine-elicited single channel currents. However, in both the α2 and α3 construct, mutating either residue that flanks Tyr 184 to its α7 counterpart restores high-affinity α-btx binding. Analogously, in α7, mutating both residues that flank Tyr 184 to the α2 or α3 counterparts abolishes high-affinity α-btx binding. Thus interaction between Tyr 184 and local residues contributes to high-affinity subtype-selective α-btx binding.« less
-
Swetha, Rayapadi G; Ramaiah, Sudha; Anbarasu, Anand
2017-08-01
Fused in sarcoma (FUS) gene encodes the RNA binding protein FUS. This gene is mapped to chromosome 16p11.2. The FUS protein binds with karyopherineβ2 (Kapβ2) through its proline/tyrosine nuclear localization signal (PY-NLS) that helps in the localization of FUS protein within the nucleus. Arginine residue in 521 position (R521) of PY-NLS plays a vital role in the binding of FUS protein with Kapβ2. Mutations in this position (R521C and R521H) are the most predominant mutations associated with amyotrophic lateral sclerosis (ALS). However, the mechanism by which these mutations lead to ALS is poorly understood. We examined the binding behaviour of the mutants FUS (R521C) and FUS (R521H) with Kapβ2 through protein-protein docking and molecular dynamics simulation. The binding patterns of mutants were compared with the binding behaviour of wild FUS-Kapβ2. Our results suggest that these mutants have relatively weak binding affinity with Kapβ2 when compared with wild FUS-Kapβ2 as indicated by the lesser number of interactions found between the mutant FUS and Kapβ2. Hence, these mutations weakens the binding and this results in the cytoplasmic mislocalization of mutant FUS; and thereby it increases the severity of ALS.
-
Gloor, Jason W; Balakrishnan, Lata; Campbell, Judith L; Bambara, Robert A
2012-08-01
In eukaryotic Okazaki fragment processing, the RNA primer is displaced into a single-stranded flap prior to removal. Evidence suggests that some flaps become long before they are cleaved, and that this cleavage involves the sequential action of two nucleases. Strand displacement characteristics of the polymerase show that a short gap precedes the flap during synthesis. Using biochemical techniques, binding and cleavage assays presented here indicate that when the flap is ∼ 30 nt long the nuclease Dna2 can bind with high affinity to the flap and downstream double strand and begin cleavage. When the polymerase idles or dissociates the Dna2 can reorient for additional contacts with the upstream primer region, allowing the nuclease to remain stably bound as the flap is further shortened. The DNA can then equilibrate to a double flap that can bind Dna2 and flap endonuclease (FEN1) simultaneously. When Dna2 shortens the flap even more, FEN1 can displace the Dna2 and cleave at the flap base to make a nick for ligation.
-
Primer design for a prokaryotic differential display RT-PCR.
Fislage, R; Berceanu, M; Humboldt, Y; Wendt, M; Oberender, H
1997-01-01
We have developed a primer set for a prokaryotic differential display of mRNA in the Enterobacteriaceae group. Each combination of ten 10mer and ten 11mer primers generates up to 85 bands from total Escherichia coli RNA, thus covering expressed sequences of a complete bacterial genome. Due to the lack of polyadenylation in prokaryotic RNA the type T11VN anchored oligonucleotides for the reverse transcriptase reaction had to be replaced with respect to the original method described by Liang and Pardee [ Science , 257, 967-971 (1992)]. Therefore, the sequences of both the 10mer and the new 11mer oligonucleotides were determined by a statistical evaluation of species-specific coding regions extracted from the EMBL database. The 11mer primers used for reverse transcription were selected for localization in the 3'-region of the bacterial RNA. The 10mer primers preferentially bind to the 5'-end of the RNA. None of the primers show homology to rRNA or other abundant small RNA species. Randomly sampled cDNA bands were checked for their bacterial origin either by re-amplification, cloning and sequencing or by re-amplification and direct sequencing with 10mer and 11mer primers after asymmetric PCR. PMID:9108168
-
Primer design for a prokaryotic differential display RT-PCR.
Fislage, R; Berceanu, M; Humboldt, Y; Wendt, M; Oberender, H
1997-05-01
We have developed a primer set for a prokaryotic differential display of mRNA in the Enterobacteriaceae group. Each combination of ten 10mer and ten 11mer primers generates up to 85 bands from total Escherichia coli RNA, thus covering expressed sequences of a complete bacterial genome. Due to the lack of polyadenylation in prokaryotic RNA the type T11VN anchored oligonucleotides for the reverse transcriptase reaction had to be replaced with respect to the original method described by Liang and Pardee [ Science , 257, 967-971 (1992)]. Therefore, the sequences of both the 10mer and the new 11mer oligonucleotides were determined by a statistical evaluation of species-specific coding regions extracted from the EMBL database. The 11mer primers used for reverse transcription were selected for localization in the 3'-region of the bacterial RNA. The 10mer primers preferentially bind to the 5'-end of the RNA. None of the primers show homology to rRNA or other abundant small RNA species. Randomly sampled cDNA bands were checked for their bacterial origin either by re-amplification, cloning and sequencing or by re-amplification and direct sequencing with 10mer and 11mer primers after asymmetric PCR.
-
DOE Office of Scientific and Technical Information (OSTI.GOV)
Verlander, P.C.; Kaporis, A.G.; Qian, L.
1994-09-01
Fanconi anemia (FA) is a genetically heterogeneous autosomal recessive disorder defined by hypersensitivity of cells to DNA cross-linking agents; a gene for complementation group C(FACC) has been cloned. Two common mutations, IVS4 +4 A{r_arrow}T and 322delG, and several rare mutations have recently been reported in affected individuals. We now report the development of amplification refractory mutation system (ARMS) assays for rapid, non-radioactive detection of these known mutations in FACC. Primer pairs specific for variant sequences were designed, with the 3{prime} terminal base of one primer matching the variant base. PCR products are separated by electrophoresis on 2.5% agarose gels; mutationsmore » are indicated by the presence of a band of a specific size. These ARMS assays can be multiplexed to allow screening for all known mutations in two PCR reactions. We have used these assays for detection of FACC mutations in affected individuals in the International Fanconi Anemia Registry (IFAR), and for carrier detection FACC families. IVS4 +4 A{r_arrow}T is the only FACC mutation found in Jewish FA patients and their families, of both Ashkenazi and Sephardic ancestry. This mutation was not found in any affected individual of non-Jewish origin. In addition, DNA samples from 1596 healthy Jewish individuals primarily of Ashkenazi ancestry were supplied to us by Dor Yeshorim. These samples, ascertained for carrier screening for Tay Sachs, cystic fibrosis, and other genetic diseases with a high frequency in the religious Jewish community served by this organization, were tested for both IVS4 +4 A{r_arrow}T and 322delG mutations; seventeen IVS4 +4 A{r_arrow}T are of Sephardic Jewish ancestry. We hypothesize that IVS4 +4 A{r_arrow}T is a very old mutation, predating the divergence of the Ashkenazi and Sephardic populations. Haplotype analysis with microsatellite markers is in progress.« less
-
Samaras, Anastasios; Madesis, Panagiotis; Karaoglanidis, George S
2016-01-01
Botrytis cinerea , is a high risk pathogen for fungicide resistance development. Pathogen' resistance to SDHIs is associated with several mutations in sdh gene. The diversity of mutations and their differential effect on cross-resistance patterns among SDHIs and the fitness of resistant strains necessitate the availability of a tool for their rapid identification. This study was initiated to develop and validate a high-resolution melting (HRM) analysis for the identification of P225H/F/L//T, N230I, and H272L/R/Y mutations. Based on the sequence of sdh B subunit of resistant and sensitive isolates, a universal primer pair was designed. The specificity of the HRM analysis primers was verified to ensure against the cross-reaction with other fungal species and its sensitivity was evaluated using concentrations of known amounts of mutant's DNA. The melting curve analysis generated nine distinct curve profiles, enabling the discrimination of all the four mutations located at codon 225, the N230I mutation, the three mutations located in codon 272, and the non-mutated isolates (isolates of wild-type sensitivity). Similar results were obtained when DNA was extracted directly from artificially inoculated strawberry fruit. The method was validated by monitoring the presence of sdh B mutations in samples of naturally infected strawberry fruits and stone fruit rootstock seedling plants showing damping-off symptoms. HRM analysis data were compared with a standard PIRA-PCR technique and an absolute agreement was observed suggesting that in both populations the H272R mutation was the predominant one, while H272Y, N230I, and P225H were detected in lower frequencies. The results of the study suggest that HRM analysis can be a useful tool for sensate, accurate, and rapid identification of several sdh B mutations in B. cinerea and it is expected to contribute in routine fungicide resistance monitoring or assessments of the effectiveness of anti-resistance strategies implemented in crops heavily treated with botryticides.
-
Samaras, Anastasios; Madesis, Panagiotis; Karaoglanidis, George S.
2016-01-01
Botrytis cinerea, is a high risk pathogen for fungicide resistance development. Pathogen’ resistance to SDHIs is associated with several mutations in sdh gene. The diversity of mutations and their differential effect on cross-resistance patterns among SDHIs and the fitness of resistant strains necessitate the availability of a tool for their rapid identification. This study was initiated to develop and validate a high-resolution melting (HRM) analysis for the identification of P225H/F/L//T, N230I, and H272L/R/Y mutations. Based on the sequence of sdhB subunit of resistant and sensitive isolates, a universal primer pair was designed. The specificity of the HRM analysis primers was verified to ensure against the cross-reaction with other fungal species and its sensitivity was evaluated using concentrations of known amounts of mutant’s DNA. The melting curve analysis generated nine distinct curve profiles, enabling the discrimination of all the four mutations located at codon 225, the N230I mutation, the three mutations located in codon 272, and the non-mutated isolates (isolates of wild-type sensitivity). Similar results were obtained when DNA was extracted directly from artificially inoculated strawberry fruit. The method was validated by monitoring the presence of sdhB mutations in samples of naturally infected strawberry fruits and stone fruit rootstock seedling plants showing damping-off symptoms. HRM analysis data were compared with a standard PIRA–PCR technique and an absolute agreement was observed suggesting that in both populations the H272R mutation was the predominant one, while H272Y, N230I, and P225H were detected in lower frequencies. The results of the study suggest that HRM analysis can be a useful tool for sensate, accurate, and rapid identification of several sdhB mutations in B. cinerea and it is expected to contribute in routine fungicide resistance monitoring or assessments of the effectiveness of anti-resistance strategies implemented in crops heavily treated with botryticides. PMID:27895633
-
Mapping Interaction Sites on Human Chemokine Receptors by Deep Mutational Scanning.
Heredia, Jeremiah D; Park, Jihye; Brubaker, Riley J; Szymanski, Steven K; Gill, Kevin S; Procko, Erik
2018-06-01
Chemokine receptors CXCR4 and CCR5 regulate WBC trafficking and are engaged by the HIV-1 envelope glycoprotein gp120 during infection. We combine a selection of human CXCR4 and CCR5 libraries comprising nearly all of ∼7000 single amino acid substitutions with deep sequencing to define sequence-activity landscapes for surface expression and ligand interactions. After consideration of sequence constraints for surface expression, known interaction sites with HIV-1-blocking Abs were appropriately identified as conserved residues following library sorting for Ab binding, validating the use of deep mutational scanning to map functional interaction sites in G protein-coupled receptors. Chemokine CXCL12 was found to interact with residues extending asymmetrically into the CXCR4 ligand-binding cavity, similar to the binding surface of CXCR4 recognized by an antagonistic viral chemokine previously observed crystallographically. CXCR4 mutations distal from the chemokine binding site were identified that enhance chemokine recognition. This included disruptive mutations in the G protein-coupling site that diminished calcium mobilization, as well as conservative mutations to a membrane-exposed site (CXCR4 residues H79 2.45 and W161 4.50 ) that increased ligand binding without loss of signaling. Compared with CXCR4-CXCL12 interactions, CCR5 residues conserved for gp120 (HIV-1 BaL strain) interactions map to a more expansive surface, mimicking how the cognate chemokine CCL5 makes contacts across the entire CCR5 binding cavity. Acidic substitutions in the CCR5 N terminus and extracellular loops enhanced gp120 binding. This study demonstrates how comprehensive mutational scanning can define functional interaction sites on receptors, and novel mutations that enhance receptor activities can be found simultaneously. Copyright © 2018 by The American Association of Immunologists, Inc.
-
Zamani, Mohammad Javad; Bihamta, Mohammad Reza; Naserian Khiabani, Behnam; Tahernezhad, Zahra; Hallajian, Mohammad Taher; Shamsi, Marzieh Varasteh
2014-01-01
Allelic diversity of HMW glutenin loci in several studies revealed that allelic combinations affect dough quality. Dx5 + Dy10 subunits are related to good baking quality and Dx2 + Dy12 are related to undesirable baking quality. One of the most regular methods to evaluate the baking quality is SDS-PAGE which is used to improve baking quality labs. Marker-assisted selection is the method which can recognize the alleles related to baking quality and this method is based on polymerase chain reaction. 10 pairs of specific primers related to Dx2, Dx2.1, Dx5, Dy10, and Dy12 subunits were used for recognizing baking quality of some wheat varieties and some mutant genotypes. Only 5 pairs of them could show the specific bands. All subunits were recognized by the primers except Dx2.1. Some of the primers were extracted from previous studies and the others were designed based on D genome subunits of wheat. SDS-PAGE method accomplished having confidence in these marker's results. To realize the effect of mutation, seed storage proteins were measured. It showed that mutation had effect on the amount of seed storage protein on the mutant seeds (which showed polymorphism).
-
Real-time PCR (qPCR) primer design using free online software.
Thornton, Brenda; Basu, Chhandak
2011-01-01
Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most commonly used fluorescent chemistries are SYBR® Green dyes and TaqMan®, Molecular Beacon or Scorpion probes. SYBR® Green is very simple to use and cost efficient. As SYBR® Green dye binds to any double-stranded DNA product, its success depends greatly on proper primer design. Many types of online primer design software are available, which can be used free of charge to design desirable SYBR® Green-based qPCR primers. This laboratory exercise is intended for those who have a fundamental background in PCR. It addresses the basic fluorescent chemistries of real-time PCR, the basic rules and pitfalls of primer design, and provides a step-by-step protocol for designing SYBR® Green-based primers with free, online software. Copyright © 2010 Wiley Periodicals, Inc.
-
Mapping Polymerization and Allostery of Hemoglobin S Using Point Mutations
Weinkam, Patrick; Sali, Andrej
2014-01-01
Hemoglobin is a complex system that undergoes conformational changes in response to oxygen, allosteric effectors, mutations, and environmental changes. Here, we study allostery and polymerization of hemoglobin and its variants by application of two previously described methods: (i) AllosMod for simulating allostery dynamics given two allosterically related input structures and (ii) a machine-learning method for dynamics- and structure-based prediction of the mutation impact on allostery (Weinkam et al. J. Mol. Biol. 2013), now applicable to systems with multiple coupled binding sites such as hemoglobin. First, we predict the relative stabilities of substates and microstates of hemoglobin, which are determined primarily by entropy within our model. Next, we predict the impact of 866 annotated mutations on hemoglobin’s oxygen binding equilibrium. We then discuss a subset of 30 mutations that occur in the presence of the sickle cell mutation and whose effects on polymerization have been measured. Seven of these HbS mutations occur in three predicted druggable binding pockets that might be exploited to directly inhibit polymerization; one of these binding pockets is not apparent in the crystal structure but only in structures generated by AllosMod. For the 30 mutations, we predict that mutation-induced conformational changes within a single tetramer tend not to significantly impact polymerization; instead, these mutations more likely impact polymerization by directly perturbing a polymerization interface. Finally, our analysis of allostery allows us to hypothesize why hemoglobin evolved to have multiple subunits and a persistent low frequency sickle cell mutation. PMID:23957820
-
Pitfalls in the molecular genetic diagnosis of Leber hereditary optic neuropathy (LHON)
DOE Office of Scientific and Technical Information (OSTI.GOV)
Johns, D.R.; Neufeld, M.J.
1993-10-01
Pathogenetic mutations in mtDNA are found in the majority of patients with Leber hereditary optic neuropathy (LHON), and molecular genetic techniques to detect them are important for diagnosis. A false-positive molecular genetic error has adverse consequences for the diagnosis of this maternally inherited disease. The authors found a number of mtDNA polymorphisms that occur adjacent to known LHON-associated mutations and that confound their molecular genetic detection. These transition mutations occur at mtDNA nt 11779 (SfaNI site loss, 11778 mutation), nt 3459 (BsaHI site loss, 3460 mutation), nt 15258 (AccI site loss, 15257 mutation), nt 14485 (mismatch primer Sau3AI site loss,more » 14484 mutation), and nt 13707 (BstNI site loss, 13708 mutation). Molecular genetic detection of the most common pathogenetic mtDNA mutations in LHON, using a single restriction enzyme, may be confounded by adjacent polymorphisms that occur with a false-positive rate of 2%-7%. 19 refs.« less
-
Ittiwut, Chupong; Pratuangdejkul, Jaturong; Supornsilchai, Vichit; Muensri, Sasipa; Hiranras, Yodporn; Sahakitrungruang, Taninee; Watcharasindhu, Suttipong; Suphapeetiporn, Kanya; Shotelersuk, Vorasuk
2017-01-01
Abnormalities of dihydrotestosterone conversion [5α-reductase deficiency: online Mendelian inheritance in man (OMIM) 607306] or actions of androgens [partial androgen insensitivity syndrome (PAIS): OMIM 312300] during the 8th-12th weeks of gestation cause varying degrees of undervirilized external genitalia in 46, XY disorders of sex development (DSD) with increased testosterone production. The objective of the study was to determine clinical and genetic characteristics of Thai patients with 46, XY DSD. A cross-sectional study was conducted in 46, XY DSD with increased testosterone production (n=43) evaluated by a human chorionic gonadotropin (hCG) stimulation test or clinical features consistent with 5α-reductase deficiency or PAIS. PCR sequencing of the entire coding regions of the SRD5A2 and AR genes was performed. Molecular modeling analysis of the androgen receptor-ligand-binding domain (AR-LBD) of a novel mutation was constructed. Mutations were found in seven patients (16.3%): five (11.6%) and two (4.7%) patients had mutations in SRD5A2 and AR, respectively. Two novel mutations, SRD5A2 c.383A>G (p.Y128C) and AR c.2176C>T (p.R726C), were identified. Dimensional structural analysis of the novel mutated AR (p.R726C) revealed that it affected the co-activator binding [binding function-3 (BF-3)], not the testosterone binding site. Short phallus length was associated with 5α-reductase deficiency. Around 16.3% of our patients with 46, XY DSD had 5α-reductase deficiency or PAIS. Two novel mutations of SRD5A2 and AR were identified. The novel mutated AR (p.R726C) might affect the co-activator binding (BF-3), not the testosterone binding site.
-
Yang, Lisha; Ijaz, Iqra; Cheng, Jingliang; Wei, Chunli; Tan, Xiaojun; Khan, Md Asaduzzaman; Fu, Xiaodong; Fu, Junjiang
2018-01-01
Choroideremia is a rare X-linked recessive inherited disorder that causes chorioretinal dystrophy leading to visual impairment in its early stages which finally causes total blindness in the affected person. It is caused due to mutations in the CHM gene. In this study, we have recruited a pedigree with choroideremia and detected a nonsense variant (c.C799T:p.R267X) in CHM of the proband (I:1). Different primer sets for amplification refractory mutation system (ARMS) were designed and PCR conditions were optimized. Then, we evaluated the sequence variant in the patient, carrier, and a fetus by using ARMS technique to identify if they inherited the pathogenic gene from parental generation; we used amniotic fluid DNA for the diagnosis of the gene in the fetus. The primer pairs, WT2+C and MT+C, amplified high specific products in different DNAs which were verified by Sanger sequencing. Based on our results, ARMS technique is fast, accurate, and reliable prenatal gene diagnostic tool to assess CHM variants. Taken together, our study indicates that ARMS technique can be used as a potential molecular tool in the diagnosis of prenatal mutation for choroideremia as well as other genetic diseases in undeveloped and developing countries, where there might be shortage of medical resources and supplies.
-
Chang, Shan; Zhang, Da-Wei; Xu, Lei; Wan, Hua; Hou, Ting-Jun; Kong, Ren
2016-11-01
RNA-binding protein with multiple splicing (RBPMS) is critical for axon guidance, smooth muscle plasticity, and regulation of cancer cell proliferation and migration. Recently, different states of the RNA-recognition motif (RRM) of RBPMS, one in its free form and another in complex with CAC-containing RNA, were determined by X-ray crystallography. In this article, the free RRM domain, its wild type complex and 2 mutant complex systems are studied by molecular dynamics (MD) simulations. Through comparison of free RRM domain and complex systems, it's found that the RNA binding facilitates stabilizing the RNA-binding interface of RRM domain, especially the C-terminal loop. Although both R38Q and T103A/K104A mutations reduce the binding affinity of RRM domain and RNA, the underlining mechanisms are different. Principal component analysis (PCA) and Molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) methods were used to explore the dynamical and recognition mechanisms of RRM domain and RNA. R38Q mutation is positioned on the homodimerization interface and mainly induces the large fluctuations of RRM domains. This mutation does not directly act on the RNA-binding interface, but some interfacial hydrogen bonds are weakened. In contrast, T103A/K104A mutations are located on the RNA-binding interface of RRM domain. These mutations obviously break most of high occupancy hydrogen bonds in the RNA-binding interface. Meanwhile, the key interfacial residues lose their favorable energy contributions upon RNA binding. The ranking of calculated binding energies in 3 complex systems is well consistent with that of experimental binding affinities. These results will be helpful in understanding the RNA recognition mechanisms of RRM domain.
-
Chang, Shan; Zhang, Da-Wei; Xu, Lei; Wan, Hua; Hou, Ting-Jun; Kong, Ren
2016-01-01
ABSTRACT RNA-binding protein with multiple splicing (RBPMS) is critical for axon guidance, smooth muscle plasticity, and regulation of cancer cell proliferation and migration. Recently, different states of the RNA-recognition motif (RRM) of RBPMS, one in its free form and another in complex with CAC-containing RNA, were determined by X-ray crystallography. In this article, the free RRM domain, its wild type complex and 2 mutant complex systems are studied by molecular dynamics (MD) simulations. Through comparison of free RRM domain and complex systems, it's found that the RNA binding facilitates stabilizing the RNA-binding interface of RRM domain, especially the C-terminal loop. Although both R38Q and T103A/K104A mutations reduce the binding affinity of RRM domain and RNA, the underlining mechanisms are different. Principal component analysis (PCA) and Molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) methods were used to explore the dynamical and recognition mechanisms of RRM domain and RNA. R38Q mutation is positioned on the homodimerization interface and mainly induces the large fluctuations of RRM domains. This mutation does not directly act on the RNA-binding interface, but some interfacial hydrogen bonds are weakened. In contrast, T103A/K104A mutations are located on the RNA-binding interface of RRM domain. These mutations obviously break most of high occupancy hydrogen bonds in the RNA-binding interface. Meanwhile, the key interfacial residues lose their favorable energy contributions upon RNA binding. The ranking of calculated binding energies in 3 complex systems is well consistent with that of experimental binding affinities. These results will be helpful in understanding the RNA recognition mechanisms of RRM domain. PMID:27592836
-
Koenig, Patrick; Lee, Chingwei V.; Sanowar, Sarah; Wu, Ping; Stinson, Jeremy; Harris, Seth F.; Fuh, Germaine
2015-01-01
The development of dual targeting antibodies promises therapies with improved efficacy over mono-specific antibodies. Here, we engineered a Two-in-One VEGF/angiopoietin 2 antibody with dual action Fab (DAF) as a potential therapeutic for neovascular age-related macular degeneration. Crystal structures of the VEGF/angiopoietin 2 DAF in complex with its two antigens showed highly overlapping binding sites. To achieve sufficient affinity of the DAF to block both angiogenic factors, we turned to deep mutational scanning in the complementarity determining regions (CDRs). By mutating all three CDRs of each antibody chain simultaneously, we were able not only to identify affinity improving single mutations but also mutation pairs from different CDRs that synergistically improve both binding functions. Furthermore, insights into the cooperativity between mutations allowed us to identify fold-stabilizing mutations in the CDRs. The data obtained from deep mutational scanning reveal that the majority of the 52 CDR residues are utilized differently for the two antigen binding function and permit, for the first time, the engineering of several DAF variants with sub-nanomolar affinity against two structurally unrelated antigens. The improved variants show similar blocking activity of receptor binding as the high affinity mono-specific antibodies against these two proteins, demonstrating the feasibility of generating a dual specificity binding surface with comparable properties to individual high affinity mono-specific antibodies. PMID:26088137
-
Gao, Chunxia; Grøtli, Morten; Eriksson, Leif A
2016-03-28
Mutations in the rearranged during transfection (RET) tyrosine kinase gene leading to gain or loss of function have been associated with the development of several human cancers and Hirschsprung's disease (HSCR). However, to what extent these mutations affect individual bio-molecular functions remains unclear. In this article, the functionally significant mutations in the RET CLD1-4 calcium-binding site which lead to HSCR, and depletion of calcium ions in the RET CLD1-4 calcium binding site, were investigated by molecular dynamics simulations--to understand the mechanistic action of the mutations or loss of calcium ions in altering the protein kinase structure, dynamics, and stability. The mutations or loss of calcium ions change the local conformation and change the free energy landscape. Specifically, the mutations and loss of calcium ions decrease the radius of gyration of the whole structure, leading to improper protein folding and GFL-GFRα contact site reduction. Furthermore, based on the most populated conformation in the wildtype MD simulations, a pharmacophore was generated by fragment docking to identify key features of the possible inhibitors targeting the calcium binding site. Overall, the findings may provide useful structural insights into the molecular mechanism underlying RET calcium-binding site mutations and assist in development of novel drugs targeting the extracellular ligand contact site of wildtype RET.
-
MutaBind estimates and interprets the effects of sequence variants on protein-protein interactions.
Li, Minghui; Simonetti, Franco L; Goncearenco, Alexander; Panchenko, Anna R
2016-07-08
Proteins engage in highly selective interactions with their macromolecular partners. Sequence variants that alter protein binding affinity may cause significant perturbations or complete abolishment of function, potentially leading to diseases. There exists a persistent need to develop a mechanistic understanding of impacts of variants on proteins. To address this need we introduce a new computational method MutaBind to evaluate the effects of sequence variants and disease mutations on protein interactions and calculate the quantitative changes in binding affinity. The MutaBind method uses molecular mechanics force fields, statistical potentials and fast side-chain optimization algorithms. The MutaBind server maps mutations on a structural protein complex, calculates the associated changes in binding affinity, determines the deleterious effect of a mutation, estimates the confidence of this prediction and produces a mutant structural model for download. MutaBind can be applied to a large number of problems, including determination of potential driver mutations in cancer and other diseases, elucidation of the effects of sequence variants on protein fitness in evolution and protein design. MutaBind is available at http://www.ncbi.nlm.nih.gov/projects/mutabind/. Published by Oxford University Press on behalf of Nucleic Acids Research 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.
-
Longworth, Michelle S; Laimins, Laimonis A
2004-04-01
The E7 oncoprotein of high-risk human papillomaviruses (HPVs) binds to and alters the action of cell cycle regulatory proteins such as members of the retinoblastoma (Rb) family of proteins as well as the histone deacetylases (HDACs). To examine the significance of the binding of E7 to HDACs in the viral life cycle, a mutational analysis of the E7 open reading frame was performed in the context of the complete HPV type 31 (HPV-31) genome. Human foreskin keratinocytes were transfected with wild-type HPV-31 genomes or HPV-31 genomes containing mutations in HDAC binding sequences as well as in the C-terminal zinc finger-like domain, and stable cell lines were isolated. All mutant genomes, except those with E7 mutations in the HDAC binding site, were found to be stably maintained extrachromosomally at an early passage following transfection. Upon further passage in culture, genomes containing mutations to the Rb binding domain as well as the zinc finger-like region quickly lost the ability to maintain episomal genomes. Genomes containing mutations abolishing E7 binding to HDACs or to Rb or mutations to the zinc finger-like motifs failed to extend the life span of transfected keratinocytes and caused cells to arrest at the same time as the untransfected keratinocytes. When induced to differentiate by suspension in methylcellulose, cells maintaining genomes with mutations in the Rb binding domain or the zinc finger-like motifs were impaired in their abilities to activate late viral functions. This study demonstrates that the interaction of E7 with HDACs and the integrity of the zinc finger-like motifs are essential for extending the life span of keratinocytes and for stable maintenance of viral genomes.
-
Direct role for the RNA polymerase domain of T7 primase in primer delivery
Zhu, Bin; Lee, Seung-Joo; Richardson, Charles C.
2010-01-01
Gene 4 protein (gp4) encoded by bacteriophage T7 contains a C-terminal helicase and an N-terminal primase domain. After synthesis of tetraribonucleotides, gp4 must transfer them to the polymerase for use as primers to initiate DNA synthesis. In vivo gp4 exists in two molecular weight forms, a 56-kDa form and the full-length 63-kDa form. The 56-kDa gp4 lacks the N-terminal Cys4 zinc-binding motif important in the recognition of primase sites in DNA. The 56-kDa gp4 is defective in primer synthesis but delivers a wider range of primers to initiate DNA synthesis compared to the 63-kDa gp4. Suppressors exist that enable the 56-kDa gp4 to support the growth of T7 phage lacking gene 4 (T7Δ4). We have identified 56-kDa DNA primases defective in primer delivery by screening for their ability to support growth of T7Δ4 phage in the presence of this suppressor. Trp69 is critical for primer delivery. Replacement of Trp69 with lysine in either the 56- or 63-kDa gp4 results in defective primer delivery with other functions unaffected. DNA primase harboring lysine at position 69 fails to stabilize the primer on DNA. Thus, a primase subdomain not directly involved in primer synthesis is involved in primer delivery. The stabilization of the primer by DNA primase is necessary for DNA polymerase to initiate synthesis. PMID:20439755
-
Knape, Kirsten; Linder, Tobias; Wolschann, Peter; Beyer, Anton; Stary-Weinzinger, Anna
2011-01-01
Pharmacological inhibition of cardiac hERG K+ channels is associated with increased risk of lethal arrhythmias. Many drugs reduce hERG current by directly binding to the channel, thereby blocking ion conduction. Mutation of two aromatic residues (F656 and Y652) substantially decreases the potency of numerous structurally diverse compounds. Nevertheless, some drugs are only weakly affected by mutation Y652A. In this study we utilize molecular dynamics simulations and docking studies to analyze the different effects of mutation Y652A on a selected number of hERG blockers. MD simulations reveal conformational changes in the binding site induced by mutation Y652A. Loss of π-π-stacking between the two aromatic residues induces a conformational change of the F656 side chain from a cavity facing to cavity lining orientation. Docking studies and MD simulations qualitatively reproduce the diverse experimentally observed modulatory effects of mutation Y652A and provide a new structural interpretation for the sensitivity differences. PMID:22194911
-
Terada, Kazuki; Yamaguchi, Hiroki; Ueki, Toshimitsu; Usuki, Kensuke; Kobayashi, Yutaka; Tajika, Kenji; Gomi, Seiji; Kurosawa, Saiko; Miyadera, Keiki; Tokura, Taichiro; Omori, Ikuko; Marumo, Atushi; Fujiwara, Yusuke; Yui, Shunsuke; Ryotokuji, Takeshi; Osaki, Yoshiki; Arai, Kunihito; Kitano, Tomoaki; Kosaka, Fumiko; Wakita, Satoshi; Tamai, Hayato; Fukuda, Takahiro; Inokuchi, Koiti
2018-01-01
TP53 gene abnormality has been reported to be an unfavorable prognostic factor in acute myeloid leukemia (AML). However, almost all studies of TP53 gene abnormality so far have been limited to mutation searches in the DNA binding domain. As there have been few reports examining both mutation and deletion over the full-length of the TP53 gene, the clinical characteristics of TP53 gene abnormality have not yet been clearly established. In this study, TP53 gene mutation was observed in 7.3% of the total 412 de novo AML cases (33 mutations in 30 cases), with mutation outside the DNA binding domain in eight cases (27%). TP53 gene deletion was observed in 3.1% of 358 cases. All cases had monoallelic deletion with TP53 gene mutation on the opposite allele. Multivariate analysis demonstrated that TP53 gene mutation in the DNA binding domain and outside the DNA binding domain was an independent poor prognostic factor for overall survival and relapse-free survival among the total cohort and it is also an unfavorable prognostic factor in FLT3-ITD-negative AML cases aged 70 years or below with intermediate cytogenetic prognosis. In stratified treatment, full-length search for TP53 gene mutation is therefore very important.
-
Gabus, Caroline; Ivanyi-Nagy, Roland; Depollier, Julien; Bucheton, Alain; Pelisson, Alain; Darlix, Jean-Luc
2006-01-01
Mobile LTR-retroelements comprising retroviruses and LTR-retrotransposons form a large part of eukaryotic genomes. Their mode of replication and abundance favour the notion that they are major actors in eukaryote evolution. The Gypsy retroelement can spread in the germ line of the fruit fly Drosophila melanogaster via both env-independent and env-dependent processes. Thus, Gypsy is both an active retrotransposon and an infectious retrovirus resembling the gammaretrovirus MuLV. However, unlike gammaretroviruses, the Gypsy Gag structural precursor is not processed into Matrix, Capsid and Nucleocapsid (NC) proteins. In contrast, it has features in common with Gag of the ancient yeast TY1 retroelement. These characteristics of Gypsy make it a very interesting model to study replication of a retroelement at the frontier between ancient retrotransposons and retroviruses. We investigated Gypsy replication using an in vitro model system and transfection of insect cells. Results show that an unstructured domain of Gypsy Gag has all the properties of a retroviral NC. This NC-like peptide forms ribonucleoparticle-like complexes upon binding Gypsy RNA and directs the annealing of primer tRNA(Lys,2) to two distinct primer binding sites (PBS) at the genome 5' and 3' ends. Only the 5' PBS is indispensable for cDNA synthesis in vitro and in Drosophila cells.
-
Gabus, Caroline; Ivanyi-Nagy, Roland; Depollier, Julien; Bucheton, Alain; Pelisson, Alain; Darlix, Jean-Luc
2006-01-01
Mobile LTR-retroelements comprising retroviruses and LTR-retrotransposons form a large part of eukaryotic genomes. Their mode of replication and abundance favour the notion that they are major actors in eukaryote evolution. The Gypsy retroelement can spread in the germ line of the fruit fly Drosophila melanogaster via both env-independent and env-dependent processes. Thus, Gypsy is both an active retrotransposon and an infectious retrovirus resembling the gammaretrovirus MuLV. However, unlike gammaretroviruses, the Gypsy Gag structural precursor is not processed into Matrix, Capsid and Nucleocapsid (NC) proteins. In contrast, it has features in common with Gag of the ancient yeast TY1 retroelement. These characteristics of Gypsy make it a very interesting model to study replication of a retroelement at the frontier between ancient retrotransposons and retroviruses. We investigated Gypsy replication using an in vitro model system and transfection of insect cells. Results show that an unstructured domain of Gypsy Gag has all the properties of a retroviral NC. This NC-like peptide forms ribonucleoparticle-like complexes upon binding Gypsy RNA and directs the annealing of primer tRNALys,2 to two distinct primer binding sites (PBS) at the genome 5′ and 3′ ends. Only the 5′ PBS is indispensable for cDNA synthesis in vitro and in Drosophila cells. PMID:17040893
-
Tighter Ligand Binding Can Compensate for Impaired Stability of an RNA-Binding Protein.
Wallis, Christopher P; Richman, Tara R; Filipovska, Aleksandra; Rackham, Oliver
2018-06-15
It has been widely shown that ligand-binding residues, by virtue of their orientation, charge, and solvent exposure, often have a net destabilizing effect on proteins that is offset by stability conferring residues elsewhere in the protein. This structure-function trade-off can constrain possible adaptive evolutionary changes of function and may hamper protein engineering efforts to design proteins with new functions. Here, we present evidence from a large randomized mutant library screen that, in the case of PUF RNA-binding proteins, this structural relationship may be inverted and that active-site mutations that increase protein activity are also able to compensate for impaired stability. We show that certain mutations in RNA-protein binding residues are not necessarily destabilizing and that increased ligand-binding can rescue an insoluble, unstable PUF protein. We hypothesize that these mutations restabilize the protein via thermodynamic coupling of protein folding and RNA binding.
-
Baugh, Loren; Le Trong, Isolde; Cerutti, David S; Gülich, Susanne; Stayton, Patrick S; Stenkamp, Ronald E; Lybrand, Terry P
2010-06-08
We have identified a distal point mutation in streptavidin that causes a 1000-fold reduction in biotin binding affinity without disrupting the equilibrium complex structure. The F130L mutation creates a small cavity occupied by a water molecule; however, all neighboring side chain positions are preserved, and protein-biotin hydrogen bonds are unperturbed. Molecular dynamics simulations reveal a reduced mobility of biotin binding residues but no observable destabilization of protein-ligand interactions. Our combined structural and computational studies suggest that the additional water molecule may affect binding affinity through an electronic polarization effect that impacts the highly cooperative hydrogen bonding network in the biotin binding pocket.
-
2014-01-01
Background Binding free energy and binding hot spots at protein-protein interfaces are two important research areas for understanding protein interactions. Computational methods have been developed previously for accurate prediction of binding free energy change upon mutation for interfacial residues. However, a large number of interrupted and unimportant atomic contacts are used in the training phase which caused accuracy loss. Results This work proposes a new method, βACV ASA , to predict the change of binding free energy after alanine mutations. βACV ASA integrates accessible surface area (ASA) and our newly defined β contacts together into an atomic contact vector (ACV). A β contact between two atoms is a direct contact without being interrupted by any other atom between them. A β contact’s potential contribution to protein binding is also supposed to be inversely proportional to its ASA to follow the water exclusion hypothesis of binding hot spots. Tested on a dataset of 396 alanine mutations, our method is found to be superior in classification performance to many other methods, including Robetta, FoldX, HotPOINT, an ACV method of β contacts without ASA integration, and ACV ASA methods (similar to βACV ASA but based on distance-cutoff contacts). Based on our data analysis and results, we can draw conclusions that: (i) our method is powerful in the prediction of binding free energy change after alanine mutation; (ii) β contacts are better than distance-cutoff contacts for modeling the well-organized protein-binding interfaces; (iii) β contacts usually are only a small fraction number of the distance-based contacts; and (iv) water exclusion is a necessary condition for a residue to become a binding hot spot. Conclusions βACV ASA is designed using the advantages of both β contacts and water exclusion. It is an excellent tool to predict binding free energy changes and binding hot spots after alanine mutation. PMID:24568581
-
Setiawan, Laurentia C; van Dort, Karel A; Rits, Maarten A N; Kootstra, Neeltje A
2016-04-01
Mutations in the cyclophilin A (CypA) binding region in the HIV-1 capsid affect their dependency on the known HIV-1 cofactor CypA and allow escape from the HIV-1 restriction factor Trim5α in human and simian cells. Here we study the effect of these mutations in the CypA binding region of capsid on cofactor binding, capsid destabilization, and viral replication in primary cells. We showed that the viral capsid with mutations in the CypA binding region (CypA-BR) interacted efficiently with CypA, but had an increased stability upon infection as compared to the wild-type capsid. Interestingly, the wild-type virus was able to infect monocyte-derived macrophages (MDM) more efficiently as compared to the CypA-BR mutant variant. The lower infectivity of the CypA-BR mutant virus in MDM was associated with lower levels of reverse transcription products. Similar to the wild-type virus, the CypA-BR mutant variant was unable to induce a strong innate response in primary macrophages. These data demonstrate that mutations in the CypA binding site of the capsid resulted in higher capsid stability and hampered infectivity in macrophages.
-
NASA Astrophysics Data System (ADS)
Kříž, Zdeněk; Adam, Jan; Mrázková, Jana; Zotos, Petros; Chatzipavlou, Thomais; Wimmerová, Michaela; Koča, Jaroslav
2014-09-01
This article focuses on designing mutations of the PA-IIL lectin from Pseudomonas aeruginosa that lead to change in specificity. Following the previous results revealing the importance of the amino acid triad 22-23-24 (so-called specificity-binding loop), saturation in silico mutagenesis was performed, with the intent of finding mutations that increase the lectin's affinity and modify its specificity. For that purpose, a combination of docking, molecular dynamics and binding free energy calculation was used. The combination of methods revealed mutations that changed the performance of the wild-type lectin and its mutants to their preferred partners. The mutation at position 22 resulted in 85 % in inactivation of the binding site, and the mutation at 23 did not have strong effects thanks to the side chain being pointed away from the binding site. Molecular dynamics simulations followed by binding free energy calculation were performed on mutants with promising results from docking, and also at those where the amino acid at position 24 was replaced for bulkier or longer polar chain. The key mutants were also prepared in vitro and their binding properties determined by isothermal titration calorimetry. Combination of the used methods proved to be able to predict changes in the lectin performance and helped in explaining the data observed experimentally.
-
Sasaki, Yohei; Fushimi, Hirotoshi; Cao, Hui; Cai, Shao-Qing; Komatsu, Katsuko
2002-12-01
The botanical origins of Chinese and Japanese Curcuma drugs were determined to be Curcuma longa, C. phaeocaulis, the Japanese population of C. zedoaria, C. kwangsiensis, C. wenyujin, and C. aromatica based on a comparison of their 18S rRNA gene and trnK gene sequences with those of six Curcuma species reported previously. Moreover, to develop a more convenient identification method, amplification-refractory mutation system (ARMS) analysis of both gene regions was performed on plants. The ARMS method for the 18S rRNA gene was established using two types of forward primers designed based on the nucleotide difference at position 234. When DNAs of four Curcuma species were used as templates, PCR amplification with either of the two primers only generated a fragment of 912 base pairs (bp). However, when DNAs of the purple-cloud type of C. kwangsiensis and C. wenyujin were used, PCR amplifications with both primers unexpectedly generated the fragment, suggesting that these two were heterozygotes. The ARMS method for the trnK gene was also established using a mixture of four types of specific reverse primers designed on the basis of base substitutions and indels among six species, and common reverse and forward primers. C. phaeocaulis or the Chinese population of C. zedoaria, the Japanese population of C. zedoaria or the purple-cloud type of C. kwangsiensis, the pubescent type of C. kwangsiensis or C. wenyujin, and C. aromatica were found to show specific fragments of 730, 185, 527 or 528, and 641 or 642 bp, respectively. All species including C. longa also showed a common fragment of 897-904 bp. Using both ARMS methods, together with information on producing areas, the identification of Curcuma plants was achieved. Moreover, the ARMS method for the trnK gene was also useful for authentication of Curcuma drugs.
-
du Plessis, Mignon; Smith, Anthony M.; Klugman, Keith P.
1998-01-01
A seminested-PCR assay, based on the amplification of the pneumococcal penicillin-binding protein 2B gene (pbp2B), was developed for the detection of penicillin-resistant and -susceptible pneumococci in cerebrospinal fluid (CSF) specimens. Species-specific primers (P5 and P6) which amplified a 682-bp conserved region of the transpeptidase-encoding region of the pbp2B gene were used. Four “resistance” primers were designed to bind to altered areas of the pbp2B gene identified in penicillin-resistant South African wild-type strains. Together with the downstream primer P6, the upstream resistance primers amplified fragments which were used to detect the presence of penicillin resistance. This system identified all 35 of the S. pneumoniae isolates evaluated, including strains of 11 different serotypes and a range of penicillin-resistant and -susceptible strains. The specificity of the assay was demonstrated by its inability to amplify DNA from other bacterial species which commonly cause meningitis. It was possible to detect pneumococcal DNA from culture-negative CSF inoculated with 2.5 pg of purified DNA or 18 CFU. Analysis of 285 CSF specimens showed that PCR detected the pneumococcus in 18 samples positive by culture, including the identification of four penicillin-resistant isolates. The positive predictive value and the negative predictive value of the assay were each 100%. PMID:9466757
-
Xu, Jing-Yuan; Zhu, Yan; Yi, Ze; Wu, Gang; Xie, Guo-Yong; Qin, Min-Jian
2018-01-01
"Wu zhu yu", which is obtained from the dried unripe fruits of Tetradium ruticarpum (A. Jussieu) T. G. Hartley, has been used as a traditional Chinese medicine for treatment of headaches, abdominal colic, and hypertension for thousands of years. The present study was designed to assess the molecular genetic diversity among 25 collected accessions of T. ruticarpum (Wu zhu yu in Chinese) from different areas of China, based on inter-primer binding site (iPBS) markers and inter-simple sequence repeat (ISSR) markers. Thirteen ISSR primers generated 151 amplification bands, of which 130 were polymorphic. Out of 165 bands that were amplified using 10 iPBS primers, 152 were polymorphic. The iPBS markers displayed a higher proportion of polymorphic loci (PPL = 92.5%) than the ISSR markers (PPL = 84.9%). The results showed that T. ruticarpum possessed high loci polymorphism and genetic differentiation occurred in this plant. The combined data of iPBS and ISSR markers scored on 25 accessions produced five clusters that approximately matched the geographic distribution of the species. The results indicated that both iPBS and ISSR markers were reliable and effective tools for analyzing the genetic diversity in T. ruticarpum. Copyright © 2018 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.
-
Efficient Mutagenesis Independent of Ligation (EMILI).
Füzik, Tibor; Ulbrich, Pavel; Ruml, Tomáš
2014-11-01
Site-directed mutagenesis is one of the most widely used techniques in life sciences. Here we describe an improved and simplified method for introducing mutations at desired sites. It consists of an inverse PCR using a plasmid template and two partially complementary primers. The synthesis step is followed by annealing of the PCR product's sticky ends, which are generated by exonuclease digestion. This method is fast, extremely efficient and cost-effective. It can be used to introduce large insertions and deletions, but also for multiple point mutations in a single step. To show the principle and to prove the efficiency of the method, we present a series of basic mutations (insertions, deletions, point mutations) on pUC19 plasmid DNA. Copyright © 2014 Elsevier B.V. All rights reserved.
-
[Clinical and genetic analysis of a patient with Treacher Collins syndrome in TCOF1 gene].
Li, Hongbo; Zhang, Xu; Li, Zhenyue; Chen, Jing; Lu, Yu; Jia, Jingjie; Yuan, Huijun; Han, Dongyi
2012-05-01
To analyze the clinical and genetic features of a patient with Treacher Collins syndrome (TCS), and identify the mutation in TCOF1 gene. The medical history was taken, and general physical examinations and otological examinations were conducted in this patient. Genomic DNA was extracted from this patient and his parents and complete TCOF1 gene coding exons were amplified by specific PCR primers. Direct sequencing was carried out to identify the mutations. The raw data was analyzed with GeneTool software and molecular biological website. We detected a heterozygous c. 1639 delAG mutation in exon 11 of TCOF1, which resulted in a truncated protein lacking normal function. This mutation is a novel mutation and the second case identified in exon 11 of in TCS. TCS patient reported in this study has unique clinical phenotype. TCOF1 gene mutation is the specific risk factor.
-
Rizzi, Giovanni; Lee, Jung-Rok; Dahl, Christina; Guldberg, Per; Dufva, Martin; Wang, Shan X; Hansen, Mikkel F
2017-09-26
Epigenetic modifications, in particular DNA methylation, are gaining increasing interest as complementary information to DNA mutations for cancer diagnostics and prognostics. We introduce a method to simultaneously profile DNA mutation and methylation events for an array of sites with single site specificity. Genomic (mutation) or bisulphite-treated (methylation) DNA is amplified using nondiscriminatory primers, and the amplicons are then hybridized to a giant magnetoresistive (GMR) biosensor array followed by melting curve measurements. The GMR biosensor platform offers scalable multiplexed detection of DNA hybridization, which is insensitive to temperature variation. The melting curve approach further enhances the assay specificity and tolerance to variations in probe length. We demonstrate the utility of this method by simultaneously profiling five mutation and four methylation sites in human melanoma cell lines. The method correctly identified all mutation and methylation events and further provided quantitative assessment of methylation density validated by bisulphite pyrosequencing.
-
DOE Office of Scientific and Technical Information (OSTI.GOV)
Gelb, Bruce D; Tartaglia, Marco; Pennacchio, Len
Diagnostic and therapeutic applications for Noonan Syndrome are described. The diagnostic and therapeutic applications are based on certain mutations in a RAS-specific guanine nucleotide exchange factor gene SOS1 or its expression product. The diagnostic and therapeutic applications are also based on certain mutations in a serine/threonine protein kinase gene RAF1 or its expression product thereof. Also described are nucleotide sequences, amino acid sequences, probes, and primers related to RAF1 or SOS1, and variants thereof, as well as host cells expressing such variants.
-
Gjetting, Torben; Petersen, Marie; Guldberg, Per; Güttler, Flemming
2001-01-01
Hyperphenylalaninemia due to a deficiency of phenylalanine hydroxylase (PAH) is an autosomal recessive disorder caused by >400 mutations in the PAH gene. Recent work has suggested that the majority of PAH missense mutations impair enzyme activity by causing increased protein instability and aggregation. In this study, we describe an alternative mechanism by which some PAH mutations may render PAH defective. Database searches were used to identify regions in the N-terminal domain of PAH with homology to the regulatory domain of prephenate dehydratase (PDH), the rate-limiting enzyme in the bacterial phenylalanine biosynthesis pathway. Naturally occurring N-terminal PAH mutations are distributed in a nonrandom pattern and cluster within residues 46–48 (GAL) and 65–69 (IESRP), two motifs highly conserved in PDH. To examine whether N-terminal PAH mutations affect the ability of PAH to bind phenylalanine at the regulatory domain, wild-type and five mutant (G46S, A47V, T63P/H64N, I65T, and R68S) forms of the N-terminal domain (residues 2–120) of human PAH were expressed as fusion proteins in Escherichia coli. Binding studies showed that the wild-type form of this domain specifically binds phenylalanine, whereas all mutations abolished or significantly reduced this phenylalanine-binding capacity. Our data suggest that impairment of phenylalanine-mediated activation of PAH may be an important disease-causing mechanism of some N-terminal PAH mutations, which may explain some well-documented genotype-phenotype discrepancies in PAH deficiency. PMID:11326337
-
One-step random mutagenesis by error-prone rolling circle amplification
Fujii, Ryota; Kitaoka, Motomitsu; Hayashi, Kiyoshi
2004-01-01
In vitro random mutagenesis is a powerful tool for altering properties of enzymes. We describe here a novel random mutagenesis method using rolling circle amplification, named error-prone RCA. This method consists of only one DNA amplification step followed by transformation of the host strain, without treatment with any restriction enzymes or DNA ligases, and results in a randomly mutated plasmid library with 3–4 mutations per kilobase. Specific primers or special equipment, such as a thermal-cycler, are not required. This method permits rapid preparation of randomly mutated plasmid libraries, enabling random mutagenesis to become a more commonly used technique. PMID:15507684
-
DOE Office of Scientific and Technical Information (OSTI.GOV)
Thomas, P.M.; Wohllk, N.; Huang, E.
1996-09-01
Familial persistent hyperinsulinemic hypoglycemia of infancy is a disorder of glucose homeostasis and is characterized by unregulated insulin secretion and profound hypoglycemia. Loss-of-function mutations in the second nucleotide-binding fold of the sulfonylurea receptor, a subunit of the pancreatic-islet {beta}-cell ATP-dependent potassium channel, has been demonstrated to be causative for persistent hyperinsulinemic hypoglycemia of infancy. We now describe three additional mutations in the first nucleotide-binding fold of the sulfonylurea-receptor gene. One point mutation disrupts the highly conserved Walker A motif of the first nucleotide-binding-fold region. The other two mutations occur in noncoding sequences required for RNA processing and are predicted tomore » disrupt the normal splicing pathway of the sulfonylurea-receptor mRNA precursor. These data suggest that both nucleotide-binding-fold regions of the sulfortylurea receptor are required for normal regulation of {beta}-cell ATP-dependent potassium channel activity and insulin secretion. 32 refs., 4 figs., 1 tab.« less
-
Kishimoto, Naoki; Onitsuka-Kishimoto, Ayano; Iga, Nozomi; Takamune, Nobutoki; Shoji, Shozo; Misumi, Shogo
2016-12-01
Human immunodeficiency virus type-1 (HIV-1) requires the packaging of human tRNA Lys3 as a primer for effective viral reverse transcription. Previously, we reported that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) suppresses the packaging efficiency of tRNA Lys3 . Although the binding of GAPDH to Pr55 gag is important for the suppression mechanism, it remains unclear which domain of GAPDH is responsible for the interaction with Pr55 gag . In this study, we show that Asp 256 , Lys 260 , Lys 263 and Glu 267 of GAPDH are important for the suppression of tRNA Lys3 packaging. Yeast two-hybrid analysis demonstrated that the C -terminal domain of GAPDH (151-335) interacts with both the matrix region (MA; 1-132) and capsid N -terminal domain (CA-NTD; 133-282). The D256R, K263E or E267R mutation of GAPDH led to the loss of the ability to bind to wild-type (WT) MA, and the D256R/K260E double mutation of GAPDH resulted in the loss of detectable binding activity to WT CA-NTD. In contrast, R58E, Q59A or Q63A of MA, and E76R or R82E of CA-NTD abrogated the interaction with the C -terminal domain of GAPDH. Multiple-substituted GAPDH mutant (D256R/K260E/K263E/E267R) retained the oligomeric formation with WT GAPDH in HIV-1 producing cells, but the incorporation level of the hetero-oligomer was decreased in viral particles. Furthermore, the viruses produced from cells expressing the D256R/K260E/K263E/E267R mutant restored tRNA Lys3 packaging efficiency because the mutant exerted a dominant negative effect by preventing WT GAPDH from binding to MA and CA-NTD and improved the reverse transcription. These findings indicate that the amino acids Asp 256 , Lys 260 , Lys 263 and Glu 267 of GAPDH is essential for the mechanism of tRNA Lys3 -packaging suppression and the D256R/K260E/K263E/E267R mutant of GAPDH acts in a dominant negative manner to suppress tRNA Lys3 packaging.
-
Koenig, Patrick; Lee, Chingwei V; Sanowar, Sarah; Wu, Ping; Stinson, Jeremy; Harris, Seth F; Fuh, Germaine
2015-09-04
The development of dual targeting antibodies promises therapies with improved efficacy over mono-specific antibodies. Here, we engineered a Two-in-One VEGF/angiopoietin 2 antibody with dual action Fab (DAF) as a potential therapeutic for neovascular age-related macular degeneration. Crystal structures of the VEGF/angiopoietin 2 DAF in complex with its two antigens showed highly overlapping binding sites. To achieve sufficient affinity of the DAF to block both angiogenic factors, we turned to deep mutational scanning in the complementarity determining regions (CDRs). By mutating all three CDRs of each antibody chain simultaneously, we were able not only to identify affinity improving single mutations but also mutation pairs from different CDRs that synergistically improve both binding functions. Furthermore, insights into the cooperativity between mutations allowed us to identify fold-stabilizing mutations in the CDRs. The data obtained from deep mutational scanning reveal that the majority of the 52 CDR residues are utilized differently for the two antigen binding function and permit, for the first time, the engineering of several DAF variants with sub-nanomolar affinity against two structurally unrelated antigens. The improved variants show similar blocking activity of receptor binding as the high affinity mono-specific antibodies against these two proteins, demonstrating the feasibility of generating a dual specificity binding surface with comparable properties to individual high affinity mono-specific antibodies. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
-
Ni, Zhong; Wang, Xiting; Zhang, Tianchen; Jin, Rong Zhong
2016-12-01
Anaplastic lymphoma kinase (ALK) has become as an important target for the treatment of various human cancers, especially non-small-cell lung cancer. A mutation, F1174C, suited in the C-terminal helix αC of ALK and distal from the small-molecule inhibitor ceritinib bound to the ATP-binding site, causes the emergence of drug resistance to ceritinib. However, the detailed mechanism for the allosteric effect of F1174C resistance mutation to ceritinib remains unclear. Here, molecular dynamics (MD) simulations and binding free energy calculations [Molecular Mechanics/Generalized Born Surface Area (MM/GBSA)] were carried out to explore the advent of drug resistance mutation in ALK. MD simulations observed that the exquisite aromatic-aromatic network formed by residues F1098, F1174, F1245, and F1271 in the wild-type ALK-ceritinib complex was disrupted by the F1174C mutation. The resulting mutation allosterically affected the conformational dynamic of P-loop and caused the upward movement of the P-loop from the ATP-binding site, thereby weakening the interaction between ceritinib and the P-loop. The subsequent MM/GBSA binding free energy calculations and decomposition analysis of binding free energy validated this prediction. This study provides mechanistic insight into the allosteric effect of F1174C resistance mutation to ceritinib in ALK and is expected to contribute to design the next-generation of ALK inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.
-
Sitarska, Ewa; Xu, Junjie; Park, Seungmee; Liu, Xiaoxia; Quade, Bradley; Stepien, Karolina; Sugita, Kyoko; Brautigam, Chad A; Sugita, Shuzo; Rizo, Josep
2017-05-06
Munc18-1 orchestrates SNARE complex assembly together with Munc13-1 to mediate neurotransmitter release. Munc18-1 binds to synaptobrevin, but the relevance of this interaction and its relation to Munc13 function are unclear. NMR experiments now show that Munc18-1 binds specifically and non-specifically to synaptobrevin. Specific binding is inhibited by a L348R mutation in Munc18-1 and enhanced by a D326K mutation designed to disrupt the 'furled conformation' of a Munc18-1 loop. Correspondingly, the activity of Munc18-1 in reconstitution assays that require Munc18-1 and Munc13-1 for membrane fusion is stimulated by the D326K mutation and inhibited by the L348R mutation. Moreover, the D326K mutation allows Munc13-1-independent fusion and leads to a gain-of-function in rescue experiments in Caenorhabditis elegans unc-18 nulls. Together with previous studies, our data support a model whereby Munc18-1 acts as a template for SNARE complex assembly, and autoinhibition of synaptobrevin binding contributes to enabling regulation of neurotransmitter release by Munc13-1.
-
Rohmah, Rista Nikmatu; Hardiyanti, Ferlany; Fatchiyah, Fatchiyah
2015-01-01
Background: RA is a systemic inflammatory disease that causes developing comorbidity conditions. This condition can cause by overproduction of pro-inflammatory cytokine. In a previous study, we have found bioactive peptide CSN1S2 from Ethawah goat milk for anti-inflammatory for repair the ileum destruction. However, the signaling transduction cascade of bioactive peptides inhibits inflammation still not clear yet. Therefore, we analyzed the signaling transduction cascade via JAK-STAT3 pathway by in vivo and in silico. Methods: The ileum was isolated DNA and amplification with specific primer. The sequence was analyzed using the Sanger sequencing method. Modeling 3D-structure was predicted by SWISS-MODEL and virtual interaction was analyzed by docking system using Pymol and Discovery Studio 4.0 software. Results: This study showed that STAT3 has target gene 480bp. The normal group and normal treating- CSN1S2 of goat milk have similarity from gene bank. Whereas, RA group had transversion mutation that the purine change into pyrimidine even cause frameshift mutation. Interestingly, after treating with the CSN1S2 protein of goat milk shows reverse to the normal acid sequence group. Based on in silico study, from eight peptides, only three peptides of CSN1S2 protein, which carried by PePT1 to enter the small intestine. The fragments are PepT1-41-NMAIHPR-47; PepT1-182-KISQYYQK-189 and PepT1-214-TNAIPYVR-221. We have found just one bioactive peptide of f182-KISQYYQK-189 is able bind to STAT3. The energy binding of f182-KISQYYQK-189 and RA-STAT3 amino acid, it was Σ = -402.43 kJ/mol and the energy binding of f182-KISQYYQK-189 and RAS-STAT3 amino acid is decreasing into Σ = -407.09 kJ/mol. Conclusion: This study suggested that the fragment 182-KISQYYQK-189 peptides from Ethawah goat milk may act as an anti-inflammatory agent via JAK-STAT3 signal transduction cascade at the cellular level. PMID:26483598
-
Luitz, Manuel P; Zacharias, Martin
2013-03-01
The endonuclease activity of the bacterial colicin 9 enzyme is controlled by the specific and high-affinity binding of immunity protein 9 (Im9). Molecular dynamics simulation studies in explicit solvent were used to investigate the free energy change associated with the mutation of two hot-spot interface residues [tyrosine (Tyr): Tyr54 and Tyr55] of Im9 to Ala. In addition, the effect of several other mutations (Leu33Ala, Leu52Ala, Val34Ala, Val37Ala, Ser48Ala, and Ile53Ala) with smaller influence on binding affinity was also studied. Good qualitative agreement of calculated free energy changes and experimental data on binding affinity of the mutations was observed. The simulation studies can help to elucidate the molecular details on how the mutations influence protein-protein binding affinity. The role of solvent and conformational flexibility of the partner proteins was studied by comparing the results in the presence or absence of solvent and with or without positional restraints. Restriction of the conformational mobility of protein partners resulted in significant changes of the calculated free energies but of similar magnitude for isolated Im9 and for the complex and therefore in only modest changes of binding free energy differences. Although the overall binding free energy change was similar for the two Tyr-Ala mutations, the physical origin appeared to be different with solvation changes contributing significantly to the Tyr55Ala mutation and to a loss of direct protein-protein interactions dominating the free energy change due to the Tyr54Ala mutation. Copyright © 2012 Wiley Periodicals, Inc.
-
Wang, Han; Wang, Yao; Guo, Wentao; Du, Bin; Huang, Xiaobing; Wu, Riping; Yang, Baoyu; Lin, Xiaoyan; Wu, Yilan
2018-01-01
Mutated anaplastic lymphoma kinase (ALK) drives the development of advanced non-small cell lung cancer (NSCLC). Most reported small-molecule inhibitors targeting the ALK domain do not display good inhibition of the G1202R solvent front mutation. The solvent front mutation was assumed to hinder drug binding. However, a different fact could be uncovered by the simulations reported in this study through a structural analog of alectinib (JH-VIII-157-02), which demonstrated potent effects against the G1202R mutation. Molecular docking, conventional molecular dynamics (MD) simulations, free energy calculations, and umbrella sampling (US) simulations were carried out to make clear the principles of the binding preferences of alectinib and JH-VIII-157-02 toward ALK WT and the ALK G1202R (ALK G1202R ) mutation. JH-VIII-157-02 has similar binding affinities to both ALK WT and ALK G1202R whereas it has has a much lower binding affinity for alectinib to ALK G1202R . Analysis of individual energy terms indicate the major variation involves the van der Waals and entropy terms. Structural analysis reveals that the conformational change of the ATP-binding glycine-rich loop was primarily responsible for the alectinib resistance, not JH-VIII-157-02. In addition, US simulations prove JH-VIII-157-02 has similar dissociative processes from both ALK WT and ALK G1202R , while alectinib is more easily dissociated from ALK G1202R than from ALK WT , thus indicating lesser residence time. Both the binding affinity and the drug residence time should be emphasized in rational drug design to overcome the G1202R solvent front mutation in ALK resistance.
-
Wang, Han; Wang, Yao; Guo, Wentao; Du, Bin; Huang, Xiaobing; Wu, Riping; Yang, Baoyu; Lin, Xiaoyan; Wu, Yilan
2018-01-01
Background Mutated anaplastic lymphoma kinase (ALK) drives the development of advanced non-small cell lung cancer (NSCLC). Most reported small-molecule inhibitors targeting the ALK domain do not display good inhibition of the G1202R solvent front mutation. The solvent front mutation was assumed to hinder drug binding. However, a different fact could be uncovered by the simulations reported in this study through a structural analog of alectinib (JH-VIII-157-02), which demonstrated potent effects against the G1202R mutation. Methods Molecular docking, conventional molecular dynamics (MD) simulations, free energy calculations, and umbrella sampling (US) simulations were carried out to make clear the principles of the binding preferences of alectinib and JH-VIII-157-02 toward ALKWT and the ALK G1202R (ALKG1202R) mutation. Results JH-VIII-157-02 has similar binding affinities to both ALKWT and ALKG1202R whereas it has has a much lower binding affinity for alectinib to ALKG1202R. Analysis of individual energy terms indicate the major variation involves the van der Waals and entropy terms. Structural analysis reveals that the conformational change of the ATP-binding glycine-rich loop was primarily responsible for the alectinib resistance, not JH-VIII-157-02. In addition, US simulations prove JH-VIII-157-02 has similar dissociative processes from both ALKWT and ALKG1202R, while alectinib is more easily dissociated from ALKG1202R than from ALKWT, thus indicating lesser residence time. Conclusion Both the binding affinity and the drug residence time should be emphasized in rational drug design to overcome the G1202R solvent front mutation in ALK resistance. PMID:29785088
-
In, K H; Asano, K; Beier, D; Grobholz, J; Finn, P W; Silverman, E K; Silverman, E S; Collins, T; Fischer, A R; Keith, T P; Serino, K; Kim, S W; De Sanctis, G T; Yandava, C; Pillari, A; Rubin, P; Kemp, J; Israel, E; Busse, W; Ledford, D; Murray, J J; Segal, A; Tinkleman, D; Drazen, J M
1997-01-01
Five lipoxygenase (5-LO) is the first committed enzyme in the metabolic pathway leading to the synthesis of the leukotrienes. We examined genomic DNA isolated from 25 normal subjects and 31 patients with asthma (6 of whom had aspirin-sensitive asthma) for mutations in the known transcription factor binding regions and the protein encoding region of the 5-LO gene. A family of mutations in the G + C-rich transcription factor binding region was identified consisting of the deletion of one, deletion of two, or addition of one zinc finger (Sp1/Egr-1) binding sites in the region 176 to 147 bp upstream from the ATG translation start site where there are normally 5 Sp1 binding motifs in tandem. Reporter gene activity directed by any of the mutant forms of the transcription factor binding region was significantly (P < 0.05) less effective than the activity driven by the wild type transcription factor binding region. Electrophoretic mobility shift assays (EMSAs) demonstrated the capacity of wild type and mutant transcription factor binding regions to bind nuclear extracts from human umbilical vein endothelial cells (HUVECs). These data are consistent with a family of mutations in the 5-LO gene that can modify reporter gene transcription possibly through differences in Sp1 and Egr-1 transactivation. PMID:9062372
-
In, K H; Asano, K; Beier, D; Grobholz, J; Finn, P W; Silverman, E K; Silverman, E S; Collins, T; Fischer, A R; Keith, T P; Serino, K; Kim, S W; De Sanctis, G T; Yandava, C; Pillari, A; Rubin, P; Kemp, J; Israel, E; Busse, W; Ledford, D; Murray, J J; Segal, A; Tinkleman, D; Drazen, J M
1997-03-01
Five lipoxygenase (5-LO) is the first committed enzyme in the metabolic pathway leading to the synthesis of the leukotrienes. We examined genomic DNA isolated from 25 normal subjects and 31 patients with asthma (6 of whom had aspirin-sensitive asthma) for mutations in the known transcription factor binding regions and the protein encoding region of the 5-LO gene. A family of mutations in the G + C-rich transcription factor binding region was identified consisting of the deletion of one, deletion of two, or addition of one zinc finger (Sp1/Egr-1) binding sites in the region 176 to 147 bp upstream from the ATG translation start site where there are normally 5 Sp1 binding motifs in tandem. Reporter gene activity directed by any of the mutant forms of the transcription factor binding region was significantly (P < 0.05) less effective than the activity driven by the wild type transcription factor binding region. Electrophoretic mobility shift assays (EMSAs) demonstrated the capacity of wild type and mutant transcription factor binding regions to bind nuclear extracts from human umbilical vein endothelial cells (HUVECs). These data are consistent with a family of mutations in the 5-LO gene that can modify reporter gene transcription possibly through differences in Sp1 and Egr-1 transactivation.
-
Sandbaken, M. G.; Culbertson, M. R.
1988-01-01
A mutational analysis of the eukaryotic elongation factor EF-1α indicates that this protein functions to limit the frequency of errors during genetic code translation. We found that both amino acid misincorporation and reading frame errors are controlled by EF-1α. In order to examine the function of this protein, the TEF2 gene, which encodes EF-1α in Saccharomyces cerevisiae, was mutagenized in vitro with hydroxylamine. Sixteen independent TEF2 alleles were isolated by their ability to suppress frameshift mutations. DNA sequence analysis identified eight different sites in the EF-1α protein that elevate the frequency of mistranslation when mutated. These sites are located in two different regions of the protein. Amino acid substitutions located in or near the GTP-binding and hydrolysis domain of the protein cause suppression of frameshift and nonsense mutations. These mutations may effect mistranslation by altering the binding or hydrolysis of GTP. Amino acid substitutions located adjacent to a putative aminoacyl-tRNA binding region also suppress frameshift and nonsense mutations. These mutations may alter the binding of aminoacyl-tRNA by EF-1α. The identification of frameshift and nonsense suppressor mutations in EF-1α indicates a role for this protein in limiting amino acid misincorporation and reading frame errors. We suggest that these types of errors are controlled by a common mechanism or closely related mechanisms. PMID:3066688
-
Brender, Jeffrey R.; Zhang, Yang
2015-01-01
The formation of protein-protein complexes is essential for proteins to perform their physiological functions in the cell. Mutations that prevent the proper formation of the correct complexes can have serious consequences for the associated cellular processes. Since experimental determination of protein-protein binding affinity remains difficult when performed on a large scale, computational methods for predicting the consequences of mutations on binding affinity are highly desirable. We show that a scoring function based on interface structure profiles collected from analogous protein-protein interactions in the PDB is a powerful predictor of protein binding affinity changes upon mutation. As a standalone feature, the differences between the interface profile score of the mutant and wild-type proteins has an accuracy equivalent to the best all-atom potentials, despite being two orders of magnitude faster once the profile has been constructed. Due to its unique sensitivity in collecting the evolutionary profiles of analogous binding interactions and the high speed of calculation, the interface profile score has additional advantages as a complementary feature to combine with physics-based potentials for improving the accuracy of composite scoring approaches. By incorporating the sequence-derived and residue-level coarse-grained potentials with the interface structure profile score, a composite model was constructed through the random forest training, which generates a Pearson correlation coefficient >0.8 between the predicted and observed binding free-energy changes upon mutation. This accuracy is comparable to, or outperforms in most cases, the current best methods, but does not require high-resolution full-atomic models of the mutant structures. The binding interface profiling approach should find useful application in human-disease mutation recognition and protein interface design studies. PMID:26506533
-
Mutations in the C-terminal fragment of DnaK affecting peptide binding.
Burkholder, W F; Zhao, X; Zhu, X; Hendrickson, W A; Gragerov, A; Gottesman, M E
1996-01-01
Escherichia coli DnaK acts as a molecular chaperone through its ATP-regulated binding and release of polypeptide substrates. Overexpressing a C-terminal fragment (CTF) of DnaK (Gly-384 to Lys-638) containing the polypeptide substrate binding domain is lethal in wild-type E. coli. This dominant-negative phenotype may result from the nonproductive binding of CTF to cellular polypeptide targets of DnaK. Mutations affecting DnaK substrate binding were identified by selecting noncytotoxic CTF mutants followed by in vitro screening. The clustering of such mutations in the three-dimensional structure of CTF suggests the model that loops L1,2 and L4,5 form a rigid core structure critical for interactions with substrate. Images Fig. 1 Fig. 2 Fig. 3 PMID:8855230
-
Defoirdt, Tom; Miyamoto, Carol M; Wood, Thomas K; Meighen, Edward A; Sorgeloos, Patrick; Verstraete, Willy; Bossier, Peter
2007-10-01
This study aimed at getting a deeper insight in the molecular mechanism by which the natural furanone (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone disrupts quorum sensing in Vibrio harveyi. Bioluminescence experiments with signal molecule receptor double mutants revealed that the furanone blocks all three channels of the V. harveyi quorum sensing system. In further experiments using mutants with mutations in the quorum sensing signal transduction pathway, the compound was found to block quorum sensing-regulated bioluminescence by interacting with a component located downstream of the Hfq protein. Furthermore, reverse transcriptase real-time polymerase chain reaction with specific primers showed that there was no effect of the furanone on luxR(Vh) mRNA levels in wild-type V. harveyi cells. In contrast, mobility shift assays showed that in the presence of the furanone, significantly lower levels of the LuxR(Vh) response regulator protein were able to bind to its target promoter sequences in wild-type V. harveyi. Finally, tests with purified LuxR(Vh) protein also showed less shifts with furanone-treated LuxR(Vh), whereas the LuxR(Vh) concentration was found not to be altered by the furanone (as determined by SDS-PAGE). Therefore, our data indicate that the furanone blocks quorum sensing in V. harveyi by rendering the quorum sensing master regulator protein LuxR(Vh) unable to bind to the promoter sequences of quorum sensing-regulated genes.
-
Krüger, Manuela; Stockinger, Herbert; Krüger, Claudia; Schüssler, Arthur
2009-01-01
* At present, molecular ecological studies of arbuscular mycorrhizal fungi (AMF) are only possible above species level when targeting entire communities. To improve molecular species characterization and to allow species level community analyses in the field, a set of newly designed AMF specific PCR primers was successfully tested. * Nuclear rDNA fragments from diverse phylogenetic AMF lineages were sequenced and analysed to design four primer mixtures, each targeting one binding site in the small subunit (SSU) or large subunit (LSU) rDNA. To allow species resolution, they span a fragment covering the partial SSU, whole internal transcribed spacer (ITS) rDNA region and partial LSU. * The new primers are suitable for specifically amplifying AMF rDNA from material that may be contaminated by other organisms (e.g., samples from pot cultures or the field), characterizing the diversity of AMF species from field samples, and amplifying a SSU-ITS-LSU fragment that allows phylogenetic analyses with species level resolution. * The PCR primers can be used to monitor entire AMF field communities, based on a single rDNA marker region. Their application will improve the base for deep sequencing approaches; moreover, they can be efficiently used as DNA barcoding primers.
-
Genetic diversity analysis of tree peony germplasm using iPBS markers.
Duan, Y B; Guo, D L; Guo, L L; Wei, D F; Hou, X G
2015-07-06
We examined the genetic diversity of 10 wild species (populations) and 55 varieties of tree peony using inter-primer binding site (iPBS) markers. From a total of 36 iPBS primers, 16 were selected based on polymorphic amplification. The number of bands amplified by each primer ranged from 9 to 19, with an average of 12.88 bands per primer. The length of bands ranged from 100 to 2000 bp, concentrated at 200 to 1800 bp. Sixteen primers amplified 206 bands in total, of which 173 bands were polymorphic with a polymorphism ratio of 83.98%. Each primer amplified 10.81 polymorphic bands on average. The data were then used to construct a phylogenetic tree using unweighted pair group method with arithmetic mean methods. Clustering analysis showed that the genetic relationships among the varieties were not only related to the genetic background or geographic origin, but also to the flowering phase, flower color, and flower type. Our data also indicated that iPBS markers were useful tools for classifying tree peony germplasms and for tree peony breeding, and the specific bands were helpful for molecular identification of tree peony varieties.
-
Kim, Seong U; Batule, Bhagwan S; Mun, Hyoyoung; Byun, Ju-Young; Shim, Won-Bo; Kim, Min-Gon
2018-02-07
We have developed a novel strategy for the colorimetric detection of PCR products by utilizing a target-specific primer modified at the 5'-end with an anti-DNAzyme sequence. A single-stranded DNAzyme sequence folds into a G-quadruplex structure with hemin and shows strong peroxidase activity. When the complementary strand binds to the DNAzyme sequence, it blocks the formation of the G-quadraduplex structure and loses its peroxidase activity. In the presence of the target gene, PCR amplification proceeds, and anti-DNAzyme sequence modified primers present in the reaction mixture form a double strand through primer extension. Therefore, it does not block the DNAzyme sequence. Further, a colorimetric signal is generated by the addition of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and H 2 O 2 at the end of the reaction. We have successfully detected a single copy of the HIV type 1 gag gene in buffer and 10 copies in human serum. The strategy developed could be used to detect DNA and RNA in complex biological samples by simple primer designing that includes DNAzyme and a DNA extended primer.
-
Di Marino, Daniele; Achsel, Tilmann; Lacoux, Caroline; Falconi, Mattia; Bagni, Claudia
2014-01-01
Mutations or deletions of FMRP, involved in the regulation of mRNA metabolism in brain, lead to the Fragile X syndrome (FXS), the most frequent form of inherited intellectual disability. A severe manifestation of the disease has been associated with the Ile304Asn mutation, located on the KH2 domain of the protein. Several hypotheses have been proposed to explain the possible molecular mechanism responsible for the drastic effect of this mutation in humans. Here, we performed a molecular dynamics simulation and show that the Ile304Asn mutation destabilizes the hydrophobic core producing a partial unfolding of two α-helices and a displacement of a third one. The affected regions show increased residue flexibility and motion. Molecular docking analysis revealed strongly reduced binding to a model single-stranded nucleic acid in agreement with known data that the two partially unfolded helices form the RNA-binding surface. The third helix, which we show here to be also affected, is involved in the PAK1 protein interaction. These two functional binding sites on the KH2 domain do not overlap spatially, and therefore, they can simultaneously bind their targets. Since the Ile304Asn mutation affects both binding sites, this may justify the severe clinical manifestation observed in the patient in which both mRNA metabolism activity and cytoskeleton remodeling would be affected.
-
Huang, M M; Arnheim, N; Goodman, M F
1992-01-01
Thermus aquaticus (Taq) DNA polymerase was used to measure the extension efficiency for all configurations of matched and mismatched base pairs at template-primer 3'-termini. The transition mispairs, A(primer).C, C.A, G.T, and T.G were extended 10(-3) to 10(-4)-fold less efficiently than their correctly paired counterparts. Relative efficiencies for extending transversion mispairs were 10(-4) to 10(-5) for T.C and T.T, about 10(-6) for A.A, and less than 10(-6) for G.A, A.G, G.G and C.C. The transversion mispair C(primer).T was extended with high efficiency, about 10(-2) compared to a correct A.T basepair. The unexpected ease of extending the C.T mismatch was not likely to have been caused by primer-template misalignment. Taq polymerase was observed to bind with similar affinities to each of the correctly paired and mispaired primer-template 3'-ends. Thus, the failure of Taq polymerase to extend mismatches efficiently appears to be an intrinsic property of the enzyme and not due to an inability to bind to 3'-terminal mispairs. For almost all of the mispairs, C.T being the exception, Taq polymerase exhibits about 100 to 1000-fold greater discrimination against mismatch extension compared to avian myeloblastosis reverse transcriptase and HIV-1 reverse transcriptase which extend most mismatched basepairs permissively. Relative mismatch extension efficiencies for Taq polymerase were measured at 45 degrees C, 55 degrees C and 70 degrees C and found to be independent of temperature. The mispair extension data should be important in designing experiments using PCR to distinguish between sequences that vary by a single nucleotide. Images PMID:1408758
-
Saingamsook, Jassada; Saeung, Atiporn; Yanola, Jintana; Lumjuan, Nongkran; Walton, Catherine; Somboon, Pradya
2017-10-10
Mutation of the voltage-gated sodium channel (VGSC) gene, or knockdown resistance (kdr) gene, is an important resistance mechanism of the dengue vector Aedes aegypti mosquitoes against pyrethroids. In many countries in Asia, a valine to glycine substitution (V1016G) and a phenylalanine to cysteine substitution (F1534C) are common in Ae. aegypti populations. The G1016 and C1534 allele frequencies have been increasing in recent years, and hence there is a need to have a simple and inexpensive tool to monitor the alleles in large scale. A multiplex PCR to detect V1016G and F1534C mutations has been developed in the current study. This study utilized primers from previous studies for detecting the mutation at position 1016 and newly designed primers to detect variants at position 1534. The PCR conditions were validated and compared with DNA sequencing using known kdr mutant laboratory strains and field collected mosquitoes. The efficacy of this method was also compared with allele-specific PCR (AS-PCR). The results of our multiplex PCR were in complete agreement with sequencing data and better than the AS-PCR. In addition, the efficiency of two non-toxic DNA staining dyes, Ultrapower™ and RedSafe™, were evaluated by comparing with ethidium bromide (EtBr) and the results were satisfactory. Our multiplex PCR method is highly reliable and useful for implementing vector surveillance in locations where the two alleles co-occur.
-
New tRNA contacts facilitate ligand binding in a Mycobacterium smegmatis T box riboswitch.
Sherwood, Anna V; Frandsen, Jane K; Grundy, Frank J; Henkin, Tina M
2018-04-10
T box riboswitches are RNA regulatory elements widely used by organisms in the phyla Firmicutes and Actinobacteria to regulate expression of amino acid-related genes. Expression of T box family genes is down-regulated by transcription attenuation or inhibition of translation initiation in response to increased charging of the cognate tRNA. Three direct contacts with tRNA have been described; however, one of these contacts is absent in a subclass of T box RNAs and the roles of several structural domains conserved in most T box RNAs are unknown. In this study, structural elements of a Mycobacterium smegmatis ileS T box riboswitch variant with an Ultrashort (US) Stem I were sequentially deleted, which resulted in a progressive decrease in binding affinity for the tRNA Ile ligand. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) revealed structural changes in conserved riboswitch domains upon interaction with the tRNA ligand. Cross-linking and mutational analyses identified two interaction sites, one between the S-turn element in Stem II and the T arm of tRNA Ile and the other between the Stem IIA/B pseudoknot and the D loop of tRNA Ile These newly identified RNA contacts add information about tRNA recognition by the T box riboswitch and demonstrate a role for the S-turn and pseudoknot elements, which resemble structural elements that are common in many cellular RNAs.
-
Functional identification and characterization of sodium binding sites in Na symporters
Loo, Donald D. F.; Jiang, Xuan; Gorraitz, Edurne; Hirayama, Bruce A.; Wright, Ernest M.
2013-01-01
Sodium cotransporters from several different gene families belong to the leucine transporter (LeuT) structural family. Although the identification of Na+ in binding sites is beyond the resolution of the structures, two Na+ binding sites (Na1 and Na2) have been proposed in LeuT. Na2 is conserved in the LeuT family but Na1 is not. A biophysical method has been used to measure sodium dissociation constants (Kd) of wild-type and mutant human sodium glucose cotransport (hSGLT1) proteins to identify the Na+ binding sites in hSGLT1. The Na1 site is formed by residues in the sugar binding pocket, and their mutation influences sodium binding to Na1 but not to Na2. For the canonical Na2 site formed by two –OH side chains, S392 and S393, and three backbone carbonyls, mutation of S392 to cysteine increased the sodium Kd by sixfold. This was accompanied by a dramatic reduction in the apparent sugar and phlorizin affinities. We suggest that mutation of S392 in the Na2 site produces a structural rearrangement of the sugar binding pocket to disrupt both the binding of the second Na+ and the binding of sugar. In contrast, the S393 mutations produce no significant changes in sodium, sugar, and phlorizin affinities. We conclude that the Na2 site is conserved in hSGLT1, the side chain of S392 and the backbone carbonyl of S393 are important in the first Na+ binding, and that Na+ binding to Na2 promotes binding to Na1 and also sugar binding. PMID:24191006
-
Expedited quantification of mutant ribosomal RNA by binary deoxyribozyme (BiDz) sensors.
Gerasimova, Yulia V; Yakovchuk, Petro; Dedkova, Larisa M; Hecht, Sidney M; Kolpashchikov, Dmitry M
2015-10-01
Mutations in ribosomal RNA (rRNA) have traditionally been detected by the primer extension assay, which is a tedious and multistage procedure. Here, we describe a simple and straightforward fluorescence assay based on binary deoxyribozyme (BiDz) sensors. The assay uses two short DNA oligonucleotides that hybridize specifically to adjacent fragments of rRNA, one of which contains a mutation site. This hybridization results in the formation of a deoxyribozyme catalytic core that produces the fluorescent signal and amplifies it due to multiple rounds of catalytic action. This assay enables us to expedite semi-quantification of mutant rRNA content in cell cultures starting from whole cells, which provides information useful for optimization of culture preparation prior to ribosome isolation. The method requires less than a microliter of a standard Escherichia coli cell culture and decreases analysis time from several days (for primer extension assay) to 1.5 h with hands-on time of ∼10 min. It is sensitive to single-nucleotide mutations. The new assay simplifies the preliminary analysis of RNA samples and cells in molecular biology and cloning experiments and is promising in other applications where fast detection/quantification of specific RNA is required. © 2015 Gerasimova et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.
-
Altered self-assembly and apatite binding of amelogenin induced by N-terminal proline mutation
Zhu, Li; Uskoković, Vuk; Le, Thuan; DenBesten, Pamela; Huang, Yulei; Habelitz, Stefan; Li, Wu
2012-01-01
Objective A single Pro-70 to Thr (p.P70T) mutation of amelogenin is known to result in hypomineralized amelogenesis imperfecta (AI). This study aims to test the hypothesis that the given mutation affects the self-assembly of amelogenin molecules and impairs their ability to conduct the growth of apatite crystals. Design Recombinant human full-length wild-type (rh174) and p.P70T mutated amelogenins were analyzed using dynamic light scattering (DLS), protein quantification assay and atomic force microscopy (AFM) before and after the binding of amelogenins to hydroxyapatite crystals. The crystal growth modulated by both amelogenins in a dynamic titration system was observed using AFM. Results As compared to rh174 amelogenin, p.P70T mutant displayed significantly increased sizes of the assemblies, higher binding affinity to apatite, and decreased crystal height. Conclusions Pro-70 plays an important structural role in the biologically relevant amelogenin self-assembly. The disturbed regularity of amelogenin nanospheres by this single mutation resulted in an increased binding to apatite and inhibited crystal growth. PMID:21081224
-
Sayre, M H; Geiduschek, E P
1990-08-01
The Bacillus subtilis bacteriophage SPO1 encodes the DNA-binding protein TF1, a homolog of the ubiquitous type II DNA-binding proteins that are components of bacterial chromatin. The known three-dimensional structure of a related protein was used in devising a scheme of site-directed mutagenesis that led to the creation of a temperature-sensitive mutation in the TF1 gene. At the nonpermissive temperature, this mutation disrupted the temporal regulation of viral protein synthesis and processing, altered the kinetics of accumulation of at least one viral transcript, and prohibited the production of infective progeny phage. We suggest that TF1 function is required to shut off the expression of several early-middle and middle viral genes and that TF1 plays a role in phage head morphogenesis. Spontaneous second-site mutations of the temperature-sensitive mutant TF1 allele that suppressed its associated phenotypes were analyzed. These suppressor mutations conferred greater amino acid sequence homology with the type II DNA-binding protein from the thermophile Bacillus stearothermophilus.
-
Bioinspired Catecholic Primers for Rigid and Ductile Dental Resin Composites.
Shin, Eeseul; Ju, Sung Won; An, Larry; Ahn, Eungjin; Ahn, Jin-Soo; Kim, Byeong-Su; Ahn, B Kollbe
2018-01-17
In the construction of dental restorative polymer composite materials, surface priming on mineral fillers is essential to improve the mechanical performance of the composites. Here we present bioinspired catechol-functionalized primers for a tougher dental resin composite containing glass fillers. The catecholic primers with different polymerizable end groups were designed and then coated on glass surfaces using a simple drop-casting or dip-coating process. The surface binding ability and possible cross-linking (coupling or chemical bridging between the glass substrate and the dental resin) of the catecholic bifunctional primers were evaluated using atomic force microscopy, contact angle measurements, and the knife shear bonding test and compared to a state-of-the-art silane-based coupling agent. Various mechanical tests including shrinkage and compression tests of the dental resin composites were also conducted. Compression tests of the composites containing the catecholic primed fillers exhibited enhanced mechanical properties, owing to the bidentate hydrogen bonding of catechol moieties to the oxide mineral surface. Furthermore, the superior biocompatibility of the primed surface was confirmed via cell attachment assay, thus providing applicability of catecholic primers for practical dental and biomedical applications.
-
Enhanced Human-Type Receptor Binding by Ferret-Transmissible H5N1 with a K193T Mutation.
Peng, Wenjie; Bouwman, Kim M; McBride, Ryan; Grant, Oliver C; Woods, Robert J; Verheije, Monique H; Paulson, James C; de Vries, Robert P
2018-05-15
All human influenza pandemics have originated from avian influenza viruses. Although multiple changes are needed for an avian virus to be able to transmit between humans, binding to human-type receptors is essential. Several research groups have reported mutations in H5N1 viruses that exhibit specificity for human-type receptors and promote respiratory droplet transmission between ferrets. Upon detailed analysis, we have found that these mutants exhibit significant differences in fine receptor specificity compared to human H1N1 and H3N2 and retain avian-type receptor binding. We have recently shown that human influenza viruses preferentially bind to α2-6-sialylated branched N-linked glycans, where the sialic acids on each branch can bind to receptor sites on two protomers of the same hemagglutinin (HA) trimer. In this binding mode, the glycan projects over the 190 helix at the top of the receptor-binding pocket, which in H5N1 would create a stearic clash with lysine at position 193. Thus, we hypothesized that a K193T mutation would improve binding to branched N-linked receptors. Indeed, the addition of the K193T mutation to the H5 HA of a respiratory-droplet-transmissible virus dramatically improves both binding to human trachea epithelial cells and specificity for extended α2-6-sialylated N-linked glycans recognized by human influenza viruses. IMPORTANCE Infections by avian H5N1 viruses are associated with a high mortality rate in several species, including humans. Fortunately, H5N1 viruses do not transmit between humans because they do not bind to human-type receptors. In 2012, three seminal papers have shown how these viruses can be engineered to transmit between ferrets, the human model for influenza virus infection. Receptor binding, among others, was changed, and the viruses now bind to human-type receptors. Receptor specificity was still markedly different compared to that of human influenza viruses. Here we report an additional mutation in ferret-transmissible H5N1 that increases human-type receptor binding. K193T seems to be a common receptor specificity determinant, as it increases human-type receptor binding in multiple subtypes. The K193T mutation can now be used as a marker during surveillance of emerging viruses to assess potential pandemic risk. Copyright © 2018 American Society for Microbiology.
-
Chung, C N; Hamaguchi, Y; Honjo, T; Kawaichi, M
1994-01-01
To map regions important for DNA binding of the mouse homologue of Suppressor of Hairless or RBP-J kappa protein, mutated mouse RBP-J kappa cDNAs were made by insertion of oligonucleotide linkers or base replacement. DNA binding assays using the mutated proteins expressed in COS cells showed that various mutations between 218 Arg and 227 Arg decreased the DNA binding activity drastically. The DNA binding activity was not affected by amino acid replacements within the integrase motif of the RBP-J kappa protein (230His-269His). Replacements between 291Arg and 323Tyr affected the DNA binding activity slightly but reproducibly. These results indicate that the region encompassing 218Arg-227Arg is critical for the DNA binding activity of RBP-J kappa. This region did not show any significant homology to motifs or domains of the previously described DNA binding proteins. Using a truncation mutant protein RBP-J kappa was shown to associate with DNA as a monomer. Images PMID:8065905
-
Yakhnin, Helen; Baker, Carol S.; Berezin, Igor; Evangelista, Michael A.; Rassin, Alisa; Romeo, Tony; Babitzke, Paul
2011-01-01
The RNA binding protein CsrA is the central component of a conserved global regulatory system that activates or represses gene expression posttranscriptionally. In every known example of CsrA-mediated translational control, CsrA binds to the 5′ untranslated region of target transcripts, thereby repressing translation initiation and/or altering the stability of the RNA. Furthermore, with few exceptions, repression by CsrA involves binding directly to the Shine-Dalgarno sequence and blocking ribosome binding. sdiA encodes the quorum-sensing receptor for N-acyl-l-homoserine lactone in Escherichia coli. Because sdiA indirectly stimulates transcription of csrB, which encodes a small RNA (sRNA) antagonist of CsrA, we further explored the relationship between sdiA and the Csr system. Primer extension analysis revealed four putative transcription start sites within 85 nucleotides of the sdiA initiation codon. Potential σ70-dependent promoters were identified for each of these primer extension products. In addition, two CsrA binding sites were predicted in the initially translated region of sdiA. Expression of chromosomally integrated sdiA′-′lacZ translational fusions containing the entire promoter and CsrA binding site regions indicates that CsrA represses sdiA expression. The results from gel shift and footprint studies demonstrate that tight binding of CsrA requires both of these sites. Furthermore, the results from toeprint and in vitro translation experiments indicate that CsrA represses translation of sdiA by directly competing with 30S ribosomal subunit binding. Thus, this represents the first example of CsrA preventing translation by interacting solely within the coding region of an mRNA target. PMID:21908661
-
Whiley, David M; Jacob, Kevin; Nakos, Jennifer; Bletchly, Cheryl; Nimmo, Graeme R; Nissen, Michael D; Sloots, Theo P
2012-06-01
Numerous real-time PCR assays have been described for detection of the influenza A H275Y alteration. However, the performance of these methods can be undermined by sequence variation in the regions flanking the codon of interest. This is a problem encountered more broadly in microbial diagnostics. In this study, we developed a modification of hybridization probe-based melting curve analysis, whereby primers are used to mask proximal mutations in the sequence targets of hybridization probes, so as to limit the potential for sequence variation to interfere with typing. The approach was applied to the H275Y alteration of the influenza A (H1N1) 2009 strain, as well as a Neisseria gonorrhoeae mutation associated with antimicrobial resistance. Assay performances were assessed using influenza A and N. gonorrhoeae strains characterized by DNA sequencing. The modified hybridization probe-based approach proved successful in limiting the effects of proximal mutations, with the results of melting curve analyses being 100% consistent with the results of DNA sequencing for all influenza A and N. gonorrhoeae strains tested. Notably, these included influenza A and N. gonorrhoeae strains exhibiting additional mutations in hybridization probe targets. Of particular interest was that the H275Y assay correctly typed influenza A strains harbouring a T822C nucleotide substitution, previously shown to interfere with H275Y typing methods. Overall our modified hybridization probe-based approach provides a simple means of circumventing problems caused by sequence variation, and offers improved detection of the influenza A H275Y alteration and potentially other resistance mechanisms.
-
Functional impact of HIV coreceptor-binding site mutations
DOE Office of Scientific and Technical Information (OSTI.GOV)
Biscone, Mark J.; Miamidian, John L.; Muchiri, John M.
2006-07-20
The bridging sheet region of the gp120 subunit of the HIV-1 Env protein interacts with the major virus coreceptors, CCR5 and CXCR4. We examined the impact of mutations in and adjacent to the bridging sheet region of an X4 tropic HIV-1 on membrane fusion and entry inhibitor susceptibility. When the V3-loop of this Env was changed so that CCR5 was used, the effects of these same mutations on CCR5 use were assayed as well. We found that coreceptor-binding site mutations had greater effects on CXCR4-mediated fusion and infection than when CCR5 was used as a coreceptor, perhaps related to differencesmore » in coreceptor affinity. The mutations also reduced use of the alternative coreceptors CCR3 and CCR8 to varying degrees, indicating that the bridging sheet region is important for the efficient utilization of both major and minor HIV coreceptors. As seen before with a primary R5 virus strain, bridging sheet mutations increased susceptibility to the CCR5 inhibitor TAK-779, which correlated with CCR5 binding efficiency. Bridging sheet mutations also conferred increased susceptibility to the CXCR4 ligand AMD-3100 in the context of the X4 tropic Env. However, these mutations had little effect on the rate of membrane fusion and little effect on susceptibility to enfuvirtide, a membrane fusion inhibitor whose activity is dependent in part on the rate of Env-mediated membrane fusion. Thus, mutations that reduce coreceptor binding and enhance susceptibility to coreceptor inhibitors can affect fusion and enfuvirtide susceptibility in an Env context-dependent manner.« less
-
Development and Characterization of Novel Bioluminecent Systems
2013-07-01
Ppy I108R. The following primers and their respective reverse compliments were used: Y447E, 5´- CT TTA ATT AAA TAC AAA GGA GAG CAG GTG GCC CCC GCT G...3´ [EcoRV] and I108R, 5´- GGA GTT GCA GTG GCG CCC GCG AAC GAC CGT TAT AAT GAA CGT-3´ [KasI] (bold represents the mutated codons, underlined...primers and their respective reverse compliments: Y447C, 5´- G AAG TCT TTA ATA AAA TAC AAA GGA TGT CAG GTG GCC CCC GCT G -3´ [PacI] and I108C, 5´- GGA
-
Arnold, Frances H.; Shao, Zhixin; Zhao, Huimin; Giver, Lorraine J.
2002-01-01
A method for in vitro mutagenesis and recombination of polynucleotide sequences based on polymerase-catalyzed extension of primer oligonucleotides is disclosed. The method involves priming template polynucleotide(s) with random-sequences or defined-sequence primers to generate a pool of short DNA fragments with a low level of point mutations. The DNA fragments are subjected to denaturization followed by annealing and further enzyme-catalyzed DNA polymerization. This procedure is repeated a sufficient number of times to produce full-length genes which comprise mutants of the original template polynucleotides. These genes can be further amplified by the polymerase chain reaction and cloned into a vector for expression of the encoded proteins.
-
Interactions of Escherichia coli σ70 within the transcription elongation complex
Daube, Shirley S.; von Hippel, Peter H.
1999-01-01
A functional transcription elongation complex can be formed without passing through a promoter by adding a complementary RNA primer and core Escherichia coli RNA polymerase in trans to an RNA-primed synthetic bubble-duplex DNA framework. This framework consists of a double-stranded DNA sequence with an internal noncomplementary DNA “bubble” containing a hybridized RNA primer. On addition of core polymerase and the requisite NTPs, the RNA primer is extended in a process that manifests most of the properties of in vitro transcription elongation. This synthetic elongation complex can also be assembled by using holo rather than core RNA polymerase, and in this study we examine the interactions and fate of the σ70 specificity subunit of the holopolymerase in the assembly process. We show that the addition of holopolymerase to the bubble-duplex construct triggers the dissociation of the sigma factor from some complexes, whereas in others the RNA oligomer is released into solution instead. These results are consistent with an allosteric competition between σ70 and the nascent RNA strand within the elongation complex and suggest that both cannot be bound to the core polymerase simultaneously. However, the dissociation of σ70 from the complex can also be stimulated by binding of the holopolymerase to the DNA bubble duplex in the absence of a hybridized RNA primer, suggesting that the binding of the core polymerase to the bubble-duplex construct also triggers a conformational change that additionally weakens the sigma–core interaction. PMID:10411885
-
Wu, Xue; Xiu, Zhilong; Li, Guohui; Luo, Bing-Hao
2013-01-01
The Asp of the RGD motif of the ligand coordinates with the β I domain metal ion dependent adhesion site (MIDAS) divalent cation, emphasizing the importance of the MIDAS in ligand binding. There appears to be two distinct groups of integrins that differ in their ligand binding affinity and adhesion ability. These differences may be due to a specific residue associated with the MIDAS, particularly the β3 residue Ala252 and corresponding Ala in the β1 integrin compared to the analogous Asp residue in the β2 and β7 integrins. Interestingly, mutations in the adjacent to MIDAS (ADMIDAS) of integrins α4β7 and αLβ2 increased the binding and adhesion abilities compared to the wild-type, while the same mutations in the α2β1, α5β1, αVβ3, and αIIbβ3 integrins demonstrated decreased ligand binding and adhesion. We introduced a mutation in the αIIbβ3 to convert this MIDAS associated Ala252 to Asp. By combination of this mutant with mutations of one or two ADMIDAS residues, we studied the effects of this residue on ligand binding and adhesion. Then, we performed molecular dynamics simulations on the wild-type and mutant αIIbβ3 integrin β I domains, and investigated the dynamics of metal ion binding sites in different integrin-RGD complexes. We found that the tendency of calculated binding free energies was in excellent agreement with the experimental results, suggesting that the variation in this MIDAS associated residue accounts for the differences in ligand binding and adhesion among different integrins, and it accounts for the conflicting results of ADMIDAS mutations within different integrins. This study sheds more light on the role of the MIDAS associated residue pertaining to ligand binding and adhesion and suggests that this residue may play a pivotal role in integrin-mediated cell rolling and firm adhesion. PMID:24116162
-
Bolado-Martínez, Enrique; Pérez-Mendoza, Ansix; Alegría-Morquecho, Francisco Monserrat; Candia-Plata, María del Carmen; Aguayo-Verdugo, María del Rosario; Alvarez-Hernández, Gerardo
2012-01-01
To perform the analysis of specific regions of the major genes associated with resistance to isoniazid or rifampin. Twenty two M. tuberculosis strains, isolated from human samples obtained in Sonora, Mexico. Specific primers for hotspots of the rpoB, katG, inhA genes and the ahpC-oxyR intergenic region were used. The purified PCR products were sequenced. Mutations in the promoter of inhA, the ahpC-oxyR region, and codon 315 of katG and in 451 or 456 codons of rpoB, were identified. Detection of mutations not previously reported requires further genotypic analysis of Mycobacterium tuberculosis isolates in Sonora.
-
Da Silva Figueiredo Celestino Gomes, Priscila; Chauvot De Beauchêne, Isaure; Panel, Nicolas; Lopez, Sophie; De Sepulveda, Paulo; Geraldo Pascutti, Pedro; Solary, Eric; Tchertanov, Luba
2016-01-01
The receptors tyrosine kinases (RTKs) for the colony stimulating factor-1, CSF-1R, and for the stem cell factor, SCFR or KIT, are important mediators of signal transduction. The abnormal function of these receptors, promoted by gain-of-function mutations, leads to their constitutive activation, associated with cancer or other proliferative diseases. A secondary effect of the mutations is the alteration of receptors’ sensitivity to tyrosine kinase inhibitors, compromising effectiveness of these molecules in clinical treatment. In particular, the mutation V560G in KIT increases its sensitivity to Imatinib, while the D816V in KIT, and D802V in CSF-1R, triggers resistance to the drug. We analyzed the Imatinib binding affinity to the native and mutated KIT (mutations V560G, S628N and D816V) and CSF-1R (mutation D802V) by using molecular dynamics simulations and energy calculations of Imatinib•target complexes. Further, we evaluated the sensitivity of the studied KIT receptors to Imatinib by measuring the inhibition of KIT phosphorylation. Our study showed that (i) the binding free energy of Imatinib to the targets is highly correlated with their experimentally measured sensitivity; (ii) the electrostatic interactions are a decisive factor affecting the binding energy; (iii) the most deleterious impact to the Imatinib sensitivity is promoted by D802V (CSF-1R) and D816V (KIT) mutations; (iv) the role of the juxtamembrane region, JMR, in the imatinib binding is accessory. These findings contribute to a better description of the mutation-induced effects alternating the targets sensitivity to Imatinib. PMID:27467080
-
Da Silva Figueiredo Celestino Gomes, Priscila; Chauvot De Beauchêne, Isaure; Panel, Nicolas; Lopez, Sophie; De Sepulveda, Paulo; Geraldo Pascutti, Pedro; Solary, Eric; Tchertanov, Luba
2016-01-01
The receptors tyrosine kinases (RTKs) for the colony stimulating factor-1, CSF-1R, and for the stem cell factor, SCFR or KIT, are important mediators of signal transduction. The abnormal function of these receptors, promoted by gain-of-function mutations, leads to their constitutive activation, associated with cancer or other proliferative diseases. A secondary effect of the mutations is the alteration of receptors' sensitivity to tyrosine kinase inhibitors, compromising effectiveness of these molecules in clinical treatment. In particular, the mutation V560G in KIT increases its sensitivity to Imatinib, while the D816V in KIT, and D802V in CSF-1R, triggers resistance to the drug. We analyzed the Imatinib binding affinity to the native and mutated KIT (mutations V560G, S628N and D816V) and CSF-1R (mutation D802V) by using molecular dynamics simulations and energy calculations of Imatinib•target complexes. Further, we evaluated the sensitivity of the studied KIT receptors to Imatinib by measuring the inhibition of KIT phosphorylation. Our study showed that (i) the binding free energy of Imatinib to the targets is highly correlated with their experimentally measured sensitivity; (ii) the electrostatic interactions are a decisive factor affecting the binding energy; (iii) the most deleterious impact to the Imatinib sensitivity is promoted by D802V (CSF-1R) and D816V (KIT) mutations; (iv) the role of the juxtamembrane region, JMR, in the imatinib binding is accessory. These findings contribute to a better description of the mutation-induced effects alternating the targets sensitivity to Imatinib.
-
Kowenz-Leutz, Elisabeth; Schuetz, Anja; Liu, Qingbin; Knoblich, Maria; Heinemann, Udo; Leutz, Achim
2016-07-01
The transcription factor CCAAT/enhancer-binding protein α (C/EBPα) regulates cell cycle arrest and terminal differentiation of neutrophils and adipocytes. Mutations in the basic leucine zipper domain (bZip) of C/EBPα are associated with acute myeloid leukemia. A widely used murine transforming C/EBPα basic region mutant (BRM2) entails two bZip point mutations (I294A/R297A). BRM2 has been discordantly described as defective for DNA binding or defective for interaction with E2F. We have separated the two BRM2 mutations to shed light on the intertwined reciprocity between C/EBPα-E2F-DNA interactions. Both, C/EBPα I294A and R297A retain transactivation capacity and interaction with E2F-DP. The C/EBPα R297A mutation destabilized DNA binding, whereas the C/EBPα I294A mutation enhanced binding to DNA. The C/EBPα R297A mutant, like BRM2, displayed enhanced interaction with E2F-DP but failed to repress E2F-dependent transactivation although both mutants were readily suppressed by E2F1 for transcription through C/EBP cis-regulatory sites. In contrast, the DNA binding enhanced C/EBPα I294A mutant displayed increased repression of E2F-DP mediated transactivation and resisted E2F-DP mediated repression. Thus, the efficient repression of E2F dependent S-phase genes and the activation of differentiation genes reside in the balanced DNA binding capacity of C/EBPα. Copyright © 2016 Elsevier B.V. All rights reserved.
-
Gao, Ling; Li, Xiao-hong; Zhao, Jian-qing; Lu, Ji-hong; Zhao, Jia-gang; Zhu, Jia-shi
2012-06-18
To examine maturational changes in expressions of Ophiocordyceps sinensis (O.sinensis) transition and transversion mutation genotypes in Cordyceps sinensis (C.sinensis) stroma. MassARRAY single nucleotide polymorphism (SNP) matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrum genotyping was used, and 8 SNP extension primers were designed based on the scattered, multiple point mutations of known sequences for the O.sinensis mutants within their internal transcribed spacer (ITS) segments. Of the extension primers, 5 (not capable of distinguishing between the 2 AT-biased genotypes) located in rDNA ITS1 and ITS2 regions: 067721-211, 067721-240, 067721-477, 067721-531 and 067721-581. The other 3 extension primers located in 5.8S rDNA region: 067740-324, 067740-328 and 067740-360, to distinguish between the 2 AT-biased genotypes. MS chromatograms at the 8 SNP sites showed dynamic alterations of mutant alleles in C.sinensis stroma. The allele for the AT-biased genotypes at 067721-211 site showed higher peak height than its GC-biased counterpart in the premature C.sinensis stroma, but disappeared with C.sinensis maturation. Chromatograms displayed not only the transition mutation alleles, but also transversion mutants. Some of the transversion mutation alleles displayed higher peak heights than those for GC- and AT-biased alleles, but their peak heights and detection rates tended to be decreased with C.sinensis maturation. When distinguishing between the 2 AT-biases, AB067744 and AB067740 genotype alleles co-existed in the premature C.sinensis stroma. The allele peak height for AB067744 genotype was greatly decreased with C.sinensis maturation, while that for AB067740 genotype increased. Co-existence of at least 5 transition and transversion mutant genotypes of O.sinensis and the dynamic changes in their expressions in C.sinensis stroma along with C.sinensis maturation may be of extreme importance in C.sinensis stroma germination and maturation, enabling C.sinensis to complete its life cycle.
-
Rational redesign of neutral endopeptidase binding to merlin and moesin proteins
Niv, Masha Y; Iida, Katsuyuki; Zheng, Rong; Horiguchi, Akio; Shen, Ruoqian; Nanus, David M
2009-01-01
Neutral endopeptidase (NEP) is a 90- to 110-kDa cell-surface peptidase that is normally expressed by numerous tissues but whose expression is lost or reduced in a variety of malignancies. The anti-tumorigenic function of NEP is mediated not only by its catalytic activity but also through direct protein–protein interactions of its cytosolic region with several binding partners, including Lyn kinase, PTEN, and ezrin/radixin/moesin (ERM) proteins. We have previously shown that mutation of the K19K20K21 basic cluster in NEPs' cytosolic region to residues QNI disrupts binding to the ERM proteins. Here we show that the ERM-related protein merlin (NF2) does not bind NEP or its cytosolic region. Using experimental data, threading, and sequence analysis, we predicted the involvement of moesin residues E159Q160 in binding to the NEP cytosolic domain. Mutation of these residues to NL (to mimic the corresponding N159L160 residues in the nonbinder merlin) disrupted moesin binding to NEP. Mutation of residues N159L160Y161K162M163 in merlin to the corresponding moesin residues resulted in NEP binding to merlin. This engineered NEP peptide–merlin interaction was diminished by the QNI mutation in NEP, supporting the role of the NEP basic cluster in binding. We thus identified the region of interaction between NEP and moesin, and engineered merlin into a NEP-binding protein. These data form the basis for further exploration of the details of NEP-ERM binding and function. PMID:19388049
-
Randáková, Alena; Dolejší, Eva; Rudajev, Vladimír; Zimčík, Pavel; Doležal, Vladimír; El-Fakahany, Esam E; Jakubík, Jan
2015-07-01
We mutated key amino acids of the human variant of the M1 muscarinic receptor that target ligand binding, receptor activation, and receptor-G protein interaction. We compared the effects of these mutations on the action of two atypical M1 functionally preferring agonists (N-desmethylclozapine and xanomeline) and two classical non-selective orthosteric agonists (carbachol and oxotremorine). Mutations of D105 in the orthosteric binding site and mutation of D99 located out of the orthosteric binding site decreased affinity of all tested agonists that was translated as a decrease in potency in accumulation of inositol phosphates and intracellular calcium mobilization. Mutation of D105 decreased the potency of the atypical agonist xanomeline more than that of the classical agonists carbachol and oxotremorine. Mutation of the residues involved in receptor activation (D71) and coupling to G-proteins (R123) completely abolished the functional responses to both classical and atypical agonists. Our data show that both classical and atypical agonists activate hM1 receptors by the same molecular switch that involves D71 in the second transmembrane helix. The principal difference among the studied agonists is rather in the way they interact with D105 in the orthosteric binding site. Furthermore, our data demonstrate a key role of D105 in xanomeline wash-resistant binding and persistent activation of hM1 by wash-resistant xanomeline. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.
-
Parry, Jesse J.; Chen, Ronald; Andrews, Rebecca; Lears, Kimberly A.
2012-01-01
G protein signaling through human somatostatin receptor subtype 2 (SSTR2) is well known, but the amino acids involved in stimulation of intracellular responses upon ligand binding have not been characterized. We constructed a series of point mutants in SSTR2 at amino acid positions 89, 139, and 140 in attempts to disrupt G protein signaling upon ligand binding. The aspartic acid changes at position 89 to either Ala, Leu, or Arg generated mutant receptors with varying expression profiles and a complete inability to bind somatostatin-14 (SST). Mutations to Asp 139 and Arg 140 also led to varying expression profiles with some mutants maintaining their affinity for SST. Mutation of Arg 140 to Ala resulted in a mutated receptor that had a Bmax and dissociation constant (Kd) similar to wild-type receptor but was still coupled to the G protein as determined in both a cAMP assay and a calcium-release assay. In contrast, mutation of Asp 139 to Asn resulted in a mutated receptor with Bmax and Kd values that were similar to wild type but was uncoupled from G protein-mediated cAMP signaling, but not calcium release. Thus, we identified mutations in SSTR2 that result in either receptor expression levels that are similar to wild type but is completely ablated for ligand binding or a receptor that maintains affinity for SST and is uncoupled from G protein-mediated cAMP signaling. PMID:22495673
-
Ngo, Son Tung; Mai, Binh Khanh; Hiep, Dinh Minh; Li, Mai Suan
2015-10-01
The binding mechanism of AC1NX476 to HIV-1 protease wild type and mutations was studied by the docking and molecular dynamics simulations. The binding free energy was calculated using the double-annihilation binding free energy method. It is shown that the binding affinity of AC1NX476 to wild type is higher than not only ritonavir but also darunavir, making AC1NX476 become attractive candidate for HIV treatment. Our theoretical results are in excellent agreement with the experimental data as the correlation coefficient between calculated and experimentally measured binding free energies R = 0.993. Residues Asp25-A, Asp29-A, Asp30-A, Ile47-A, Gly48-A, and Val50-A from chain A, and Asp25-B from chain B play a crucial role in the ligand binding. The mutations were found to reduce the receptor-ligand interaction by widening the binding cavity, and the binding propensity is mainly driven by the van der Waals interaction. Our finding may be useful for designing potential drugs to combat with HIV. © 2015 John Wiley & Sons A/S.
-
Leal, Walter S.; Ishida, Yuko; Pelletier, Julien; Xu, Wei; Rayo, Josep; Xu, Xianzhong; Ames, James B.
2009-01-01
Background The navel orangeworm, Amyelois transitella Walker (Lepidoptera: Pyralidae), is the most serious insect pest of almonds and pistachios in California for which environmentally friendly alternative methods of control — like pheromone-based approaches — are highly desirable. Some constituents of the sex pheromone are unstable and could be replaced with parapheromones, which may be designed on the basis of molecular interaction of pheromones and pheromone-detecting olfactory proteins. Methodology By analyzing extracts from olfactory and non-olfactory tissues, we identified putative olfactory proteins, obtained their N-terminal amino acid sequences by Edman degradation, and used degenerate primers to clone the corresponding cDNAs by SMART RACE. Additionally, we used degenerate primers based on conserved sequences of known proteins to fish out other candidate olfactory genes. We expressed the gene encoding a newly identified pheromone-binding protein, which was analyzed by circular dichroism, fluorescence, and nuclear magnetic resonance, and used in a binding assay to assess affinity to pheromone components. Conclusion We have cloned nine cDNAs encoding olfactory proteins from the navel orangeworm, including two pheromone-binding proteins, two general odorant-binding proteins, one chemosensory protein, one glutathione S-transferase, one antennal binding protein X, one sensory neuron membrane protein, and one odorant receptor. Of these, AtraPBP1 is highly enriched in male antennae. Fluorescence, CD and NMR studies suggest a dramatic pH-dependent conformational change, with high affinity to pheromone constituents at neutral pH and no binding at low pH. PMID:19789654
-
The role of the DNA sliding clamp in Okazaki fragment maturation in archaea and eukaryotes.
Beattie, Thomas R; Bell, Stephen D
2011-01-01
Efficient processing of Okazaki fragments generated during discontinuous lagging-strand DNA replication is critical for the maintenance of genome integrity. In eukaryotes, a number of enzymes co-ordinate to ensure the removal of initiating primers from the 5'-end of each fragment and the generation of a covalently linked daughter strand. Studies in eukaryotic systems have revealed that the co-ordination of DNA polymerase δ and FEN-1 (Flap Endonuclease 1) is sufficient to remove the majority of primers. Other pathways such as that involving Dna2 also operate under certain conditions, although, notably, Dna2 is not universally conserved between eukaryotes and archaea, unlike the other core factors. In addition to the catalytic components, the DNA sliding clamp, PCNA (proliferating-cell nuclear antigen), plays a pivotal role in binding and co-ordinating these enzymes at sites of lagging-strand replication. Structural studies in eukaryotic and archaeal systems have revealed that PCNA-binding proteins can adopt different conformations when binding PCNA. This conformational malleability may be key to the co-ordination of these enzymes' activities.
-
Geuens, Thomas; De Winter, Vicky; Rajan, Nicholas; Achsel, Tilmann; Mateiu, Ligia; Almeida-Souza, Leonardo; Asselbergh, Bob; Bouhy, Delphine; Auer-Grumbach, Michaela; Bagni, Claudia; Timmerman, Vincent
2017-01-11
The small heat shock protein HSPB1 (Hsp27) is an ubiquitously expressed molecular chaperone able to regulate various cellular functions like actin dynamics, oxidative stress regulation and anti-apoptosis. So far disease causing mutations in HSPB1 have been associated with neurodegenerative diseases such as distal hereditary motor neuropathy, Charcot-Marie-Tooth disease and amyotrophic lateral sclerosis. Most mutations in HSPB1 target its highly conserved α-crystallin domain, while other mutations affect the C- or N-terminal regions or its promotor. Mutations inside the α-crystallin domain have been shown to enhance the chaperone activity of HSPB1 and increase the binding to client proteins. However, the HSPB1-P182L mutation, located outside and downstream of the α-crystallin domain, behaves differently. This specific HSPB1 mutation results in a severe neuropathy phenotype affecting exclusively the motor neurons of the peripheral nervous system. We identified that the HSPB1-P182L mutant protein has a specifically increased interaction with the RNA binding protein poly(C)binding protein 1 (PCBP1) and results in a reduction of its translational repressive activity. RNA immunoprecipitation followed by RNA sequencing on mouse brain lead to the identification of PCBP1 mRNA targets. These targets contain larger 3'- and 5'-UTRs than average and are enriched in an RNA motif consisting of the CTCCTCCTCCTCC consensus sequence. Interestingly, next to the clear presence of neuronal transcripts among the identified PCBP1 targets we identified known genes associated with hereditary peripheral neuropathies and hereditary spastic paraplegias. We therefore conclude that HSPB1 can mediate translational repression through interaction with an RNA binding protein further supporting its role in neurodegenerative disease.
-
Zeyer, Karina A; Reinhardt, Dieter P
2015-01-01
Fibrillins are the major components of microfibrils in the extracellular matrix of elastic and non-elastic tissues. They are multi-domain proteins, containing primarily calcium binding epidermal growth factor-like (cbEGF) domains and 8-cysteine/transforming growth factor-beta binding protein-like (TB) domains. Mutations in the fibrillin-1 gene give rise to Marfan syndrome, a connective tissue disorder with clinical complications in the cardiovascular, skeletal, ocular and other organ systems. Here, we review the consequences of engineered Marfan syndrome mutations in fibrillin-1 at the protein, cellular and organismal levels. Representative point mutations associated with Marfan syndrome in affected individuals have been introduced and analyzed in recombinant fibrillin-1 fragments. Those mutations affect fibrillin-1 on a structural and functional level. Mutations which impair folding of cbEGF domains can affect protein trafficking. Protein folding disrupted by some mutations can lead to defective secretion in mutant fibrillin-1 fragments, whereas fragments with other Marfan mutations are secreted normally. Many Marfan mutations render fibrillin-1 more susceptible to proteolysis. There is also evidence that some mutations affect heparin binding. Few mutations have been further analyzed in mouse models. An extensively studied mouse model of Marfan syndrome expresses mouse fibrillin-1 with a missense mutation (p.C1039G). The mice display similar characteristics to human patients with Marfan syndrome. Overall, the analyses of engineered mutations leading to Marfan syndrome provide important insights into the pathogenic molecular mechanisms exerted by mutated fibrillin-1. Copyright © 2015 Elsevier B.V. All rights reserved.
-
Spassov, Velin Z; Yan, Lisa
2013-04-01
Understanding the effects of mutation on pH-dependent protein binding affinity is important in protein design, especially in the area of protein therapeutics. We propose a novel method for fast in silico mutagenesis of protein-protein complexes to calculate the effect of mutation as a function of pH. The free energy differences between the wild type and mutants are evaluated from a molecular mechanics model, combined with calculations of the equilibria of proton binding. The predicted pH-dependent energy profiles demonstrate excellent agreement with experimentally measured pH-dependency of the effect of mutations on the dissociation constants for the complex of turkey ovomucoid third domain (OMTKY3) and proteinase B. The virtual scanning mutagenesis identifies all hotspots responsible for pH-dependent binding of immunoglobulin G (IgG) to neonatal Fc receptor (FcRn) and the results support the current understanding of the salvage mechanism of the antibody by FcRn based on pH-selective binding. The method can be used to select mutations that change the pH-dependent binding profiles of proteins and guide the time consuming and expensive protein engineering experiments. As an application of this method, we propose a computational strategy to search for mutations that can alter the pH-dependent binding behavior of IgG to FcRn with the aim of improving the half-life of therapeutic antibodies in the target organism. Copyright © 2013 Wiley Periodicals, Inc.
-
Tischer, Alexander; Campbell, James C.; Machha, Venkata R.; ...
2015-12-16
Unusually large von Willebrand factor (VWF), the first responder to vascular injury in primary hemostasis, is designed to capture platelets under the high shear stress of rheological blood flow. In type 2M von Willebrand disease, two rare mutations (G1324A and G1324S) within the platelet GPIbα binding interface of the VWF A1 domain impair the hemostatic function of VWF. We investigate structural and conformational effects of these mutations on the A1 domain's efficacy to bind collagen and adhere platelets under shear flow. These mutations enhance the thermodynamic stability, reduce the rate of unfolding, and enhance the A1 domain's resistance to limitedmore » proteolysis. Collagen binding affinity is not significantly affected indicating that the primary stabilizing effect of these mutations is to diminish the platelet binding efficiency under shear flow. The better stability stems from the steric consequences of adding a side chain (G1324A) and additionally a hydrogen bond (G1324S) to His 1322 across the β2-β3 hairpin in the GPIbα binding interface, which restrains the conformational degrees of freedom and the overall flexibility of the native state. These studies reveal a novel rheological strategy in which the incorporation of a single glycine within the GPIbα binding interface of normal VWF enhances the probability of local unfolding that enables the A1 domain to conformationally adapt to shear flow while maintaining its overall native structure.« less
-
Srivastava, Gaurava; Tripathi, Shubhandra; Kumar, Akhil; Sharma, Ashok
2017-07-01
Multi drug resistant tuberculosis is a major threat for mankind. Resistance against Isoniazid (INH), targeting MtKatG protein, is one of the most commonly occurring resistances in MDR TB strains. S315T-MtKatG mutation is widely reported for INH resistance. Despite having knowledge about the mechanism of INH, exact binding site of INH to MtKatG is still uncertain and proposed to have three presumable binding sites (site-1, site-2, and site-3). In the current study docking, molecular dynamics simulation, binding free energy estimation, principal component analysis and free energy landscape analysis were performed to get molecular level details of INH binding site on MtKatG, and to probe the effect of S315T mutation on INH binding. Molecular docking and MD analysis suggested site-1 as active binding site of INH, where the effects of S315T mutation were observed on both access tunnel as well as molecular interaction between INH and its neighboring residues. MMPBSA also supported site-1 as potential binding site with lowest binding energy of -44.201 kJ/mol. Moreover, PCA and FEL revealed that S315T mutation not only reduces the dimension of heme access tunnel but also showed that extra methyl group at 315 position altered heme cavity, enforcing heme group distantly from INH, and thus preventing INH activation. The present study not only investigated the active binding site of INH but also provides a new insight about the conformational changes in the binding site of S315T-MtKatG. Copyright © 2017 Elsevier Ltd. All rights reserved.
-
A novel large deletion mutation of FERMT1 gene in a Chinese patient with Kindler syndrome.
Gao, Ying; Bai, Jin-li; Liu, Xiao-yan; Qu, Yu-jin; Cao, Yan-yan; Wang, Jian-cai; Jin, Yu-wei; Wang, Hong; Song, Fang
2015-11-01
Kindler syndrome (KS; OMIM 173650) is a rare autosomal recessive skin disorder, which results in symptoms including blistering, epidermal atrophy, increased risk of cancer, and poor wound healing. The majority of mutations of the disease-determining gene (FERMT1 gene) are single nucleotide substitutions, including missense mutations, nonsense mutations, etc. Large deletion mutations are seldom reported. To determine the mutation in the FERMT1 gene associated with a 7-year-old Chinese patient who presented clinical manifestation of KS, we performed direct sequencing of all the exons of FERMT1 gene. For the exons 2-6 without amplicons, we analyzed the copy numbers using quantitative real-time polymerase chain reaction (qRT-PCR) with specific primers. The deletion breakpoints were sublocalized and the range of deletion was confirmed by PCR and direct sequencing. In this study, we identified a new 17-kb deletion mutation spanning the introns 1-6 of FERMT1 gene in a Chinese patient with severe KS phenotypes. Her parents were carriers of the same mutation. Our study reported a newly identified large deletion mutation of FERMT1 gene involved in KS, which further enriched the mutation spectrum of the FERMT1 gene.
-
Sultana, Afia; Sridhar, Mittanamalli S; Jagannathan, Aparna; Balasubramanian, Dorairajan; Kannabiran, Chitra; Klintworth, Gordon K
2003-12-22
Macular corneal dystrophy (MCD) is an autosomal recessive disorder characterized by progressive central haze, confluent punctate opacities and abnormal deposits in the cornea. It is caused by mutations in the carbohydrate sulfotransferase-6 (CHST6) gene, encoding corneal N-acetyl glucosamine-6-O-sulfotransferase (C-GlcNAc-6-ST). We screened the CHST6 gene for mutations in Indian families with MCD, in order to determine the range of pathogenic mutations. Genomic DNA was isolated from peripheral blood leukocytes of patients with MCD and normal controls. The coding regions of the CHST6 gene were amplified using three pairs of primers and amplified products were directly sequenced. We identified 22 (5 nonsense, 5 frameshift, 2 insertion, and 10 missense) mutations in 36 patients from 31 families with MCD, supporting the conclusion that loss of function of this gene is responsible for this corneal disease. Seventeen of these mutations are novel. These data highlight the allelic heterogeneity of macular corneal dystrophy in Indian patients.
-
NASA Technical Reports Server (NTRS)
Singer, M. S.; Oliveira, L.; Vriend, G.; Shepherd, G. M.
1995-01-01
A family of G-protein-coupled receptors is believed to mediate the recognition of odor molecules. In order to identify potential ligand-binding residues, we have applied correlated mutation analysis to receptor sequences from the rat. This method identifies pairs of sequence positions where residues remain conserved or mutate in tandem, thereby suggesting structural or functional importance. The analysis supported molecular modeling studies in suggesting several residues in positions that were consistent with ligand-binding function. Two of these positions, dominated by histidine residues, may play important roles in ligand binding and could confer broad specificity to mammalian odor receptors. The presence of positive (overdominant) selection at some of the identified positions provides additional evidence for roles in ligand binding. Higher-order groups of correlated residues were also observed. Each group may interact with an individual ligand determinant, and combinations of these groups may provide a multi-dimensional mechanism for receptor diversity.
-
Hanski, E; Caparon, M
1992-07-01
Binding to fibronectin has been suggested to play an important role in adherence of the group A streptococcus Streptococcus pyrogenes to host epithelial cells; however, the identity of the streptococcal fibronectin receptor has been elusive. Here we demonstrate that the fibronectin-binding property of S. pyogenes is mediated by protein F, a bacterial surface protein that binds fibronectin at high affinity. The gene encoding protein F (prtF) produced a functional fibronectin-binding protein in Escherichia coli. Insertional mutagenesis of the cloned gene generated a mutation that resulted in the loss of fibronectin-binding activity. When this mutation was introduced into the S. pyrogenes chromosome by homologous recombination with the wild-type allele, the resulting strains no longer produced protein F and lost their ability to bind fibronectin. The mutation could be complemented by prtF introduced on a plasmid. Mutants lacking protein F had a much lower capacity to adhere to respiratory epithelial cells. These results demonstrate that protein F is an important adhesin of S. pyogenes.
-
Impact of gamma rays on the Phaffia rhodozyma genome revealed by RAPD-PCR.
Najafi, N; Hosseini, Ramin; Ahmadi, Ar
2011-12-01
Phaffia rhodozyma is a red yeast which produces astaxanthin as the major carotenoid pigment. Astaxanthin is thought to reduce the incidence of cancer and degenerative diseases in man. It also enhances the immune response and acts as a free-radical quencher, a precursor of vitamin A, or a pigment involved in the visual attraction of animals as mating partners. The impact of gamma irradiation was studied on the Phaffia rhodozyma genome. Ten mutant strains, designated Gam1-Gam10, were obtained using gamma irradiation. Ten decamer random amplified polymorphic DNA (RAPD) primers were employed to assess genetic changes. Nine primers revealed scorable polymorphisms and a total of 95 band positions were scored; amongst which 38 bands (37.5%) were polymorphic. Primer F with 3 bands and primer J20 with 13 bands produced the lowest and the highest number of bands, respectively. Primer A16 produced the highest number of polymorphic bands (70% polymorphism) and primer F showed the lowest number of polymorphic bands (0% polymorphism). Genetic distances were calculated using Jaccard's coefficient and the UPGMA method. A dendrogram was created using SPSS (version 11.5) and the strains were clustered into four groups. RAPD markers could distinguish between the parental and the mutant strains of P. rhodozyma. RAPD technique showed that some changes had occurred in the genome of the mutated strains. This technique demonstrated the capability to differentiate between the parental and the mutant strains.
-
Willjes, Gesche; Mahrhold, Stefan; Strotmeier, Jasmin; Eichner, Timo; Rummel, Andreas; Binz, Thomas
2013-06-04
Botulinum neurotoxins (BoNTs) block neurotransmitter release by proteolyzing SNARE proteins in peripheral nerve terminals. Entry into neurons occurs subsequent to interaction with gangliosides and a synaptic vesicle protein. Isoforms I and II of synaptotagmin were shown to act as protein receptors for two of the seven BoNT serotypes, BoNT/B and BoNT/G, and for mosaic-type BoNT/DC. BoNT/B and BoNT/G exhibit a homologous binding site for synaptotagmin whose interacting part adopts helical structure upon binding to BoNT/B. Whereas the BoNT/B-synaptotagmin-II interaction has been elucidated in molecular detail, corresponding information about BoNT/G is lacking. Here we systematically mutated the synaptotagmin binding site in BoNT/G and performed a comparative binding analysis with mutants of the cell binding subunit of BoNT/B. The results suggest that synaptotagmin takes the same overall orientation in BoNT/B and BoNT/G governed by the strictly conserved central parts of the toxins' binding site. The surrounding nonconserved areas differently contribute to receptor binding. Reciprocal mutations Y1186W and L1191Y increased the level of binding of BoNT/G approximately to the level of BoNT/B affinity, suggesting a similar synaptotagmin-bound state. The effects of the mutations were confirmed by studying the activity of correspondingly mutated full-length BoNTs. On the basis of these data, molecular modeling experiments were employed to reveal an atomistic model of BoNT/G-synaptotagmin recognition. These data suggest a reduced length and/or a bend in the C-terminal part of the synaptotagmin helix that forms upon contact with BoNT/G as compared with BoNT/B and are in agreement with the data of the mutational analyses.
-
Polevoda, Bogdan; Joseph, Rebecca; Friedman, Alan E.; Bennett, Ryan P.; Greiner, Rebecca; De Zoysa, Thareendra; Stewart, Ryan A.; Smith, Harold C.
2017-01-01
APOBEC3G (A3G) belongs to the AID/APOBEC protein family of cytidine deaminases (CDA) that bind to nucleic acids. A3G mutates the HIV genome by deamination of dC to dU, leading to accumulation of virus-inactivating mutations. Binding to cellular RNAs inhibits A3G binding to substrate single-stranded (ss) DNA and CDA activity. Bulk RNA and substrate ssDNA bind to the same three A3G tryptic peptides (amino acids 181–194, 314–320, and 345–374) that form parts of a continuously exposed protein surface extending from the catalytic domain in the C terminus of A3G to its N terminus. We show here that the A3G tyrosines 181 and 315 directly cross-linked ssDNA. Binding experiments showed that a Y315A mutation alone significantly reduced A3G binding to both ssDNA and RNA, whereas Y181A and Y182A mutations only moderately affected A3G nucleic acid binding. Consistent with these findings, the Y315A mutant exhibited little to no deaminase activity in an Escherichia coli DNA mutator reporter, whereas Y181A and Y182A mutants retained ∼50% of wild-type A3G activity. The Y315A mutant also showed a markedly reduced ability to assemble into viral particles and had reduced antiviral activity. In uninfected cells, the impaired RNA-binding capacity of Y315A was evident by a shift of A3G from high-molecular-mass ribonucleoprotein complexes to low-molecular-mass complexes. We conclude that Tyr-315 is essential for coordinating ssDNA interaction with or entry to the deaminase domain and hypothesize that RNA bound to Tyr-315 may be sufficient to competitively inhibit ssDNA deaminase-dependent antiviral activity. PMID:28381554
-
The androgen receptor gene mutations database.
Gottlieb, B; Trifiro, M; Lumbroso, R; Vasiliou, D M; Pinsky, L
1996-01-01
The current version of the androgen receptor (AR) gene mutations database is described. We have added (if available) data on the androgen binding phenotype of the mutant AR, the clinical phenotype of the affected persons, the family history and whether the pathogenicity of a mutation has been proven. Exonic mutations are now listed in 5'-->3' sequence regardless of type and single base pair changes are presented in codon context. Splice site and intronic mutations are listed separately. The database has allowed us to substantiate and amplify the observation of mutational hot spots within exons encoding the AR androgen binding domain. The database is available from EML (ftp://www.ebi.ac.uk/pub/databases/androgen) or as a Macintosh Filemaker file (MC33@musica.mcgill.ca).
-
Wile, Daryl J; Agarwal, Pankaj A; Schulzer, Michael; Mak, Edwin; Dinelle, Katherine; Shahinfard, Elham; Vafai, Nasim; Hasegawa, Kazuko; Zhang, Jing; McKenzie, Jessamyn; Neilson, Nicole; Strongosky, Audrey; Uitti, Ryan J; Guttman, Mark; Zabetian, Cyrus P; Ding, Yu-Shin; Adam, Mike; Aasly, Jan; Wszolek, Zbigniew K; Farrer, Matthew; Sossi, Vesna; Stoessl, A Jon
2017-05-01
People with Parkinson's disease can show premotor neurochemical changes in the dopaminergic and non-dopaminergic systems. Using PET, we assessed whether dopaminergic and serotonin transporter changes are similar in LRRK2 mutation carriers with Parkinson's disease and individuals with sporadic Parkinson's disease, and whether LRRK2 mutation carriers without motor symptoms show PET changes. We did two cross-sectional PET studies at the Pacific Parkinson's Research Centre in Vancouver, BC, Canada. We included LRRK2 mutation carriers with or without manifest Parkinson's disease, people with sporadic Parkinson's disease, and age-matched healthy controls, all aged 18 years or older. People with Parkinson's disease were diagnosed by a neurologist with movement disorder training, in accordance with the UK Parkinson's Disease Society Brain Bank criteria. LRRK2 carrier status was confirmed by bidirectional Sanger sequencing. In the first study, LRRK2 mutation carriers with or without manifest Parkinson's disease who were referred for investigation between July, 1999, and January, 2012, were scanned with PET tracers for the membrane dopamine transporter, and dopamine synthesis and storage ( 18 F-6-fluoro-L-dopa; 18 F-FDOPA). We compared findings with those in people with sporadic Parkinson's disease and age-matched healthy controls. In the second study, distinct groups of LRRK2 mutation carriers, individuals with sporadic Parkinson's disease, and age-matched healthy controls seen from November, 2012, to May, 2016, were studied with tracers for the serotonin transporter and vesicular monoamine transporter 2 (VMAT2). Striatal dopamine transporter binding, VMAT2 binding, 18 F-FDOPA uptake, and serotonin transporter binding in multiple brain regions were compared by ANCOVA, adjusted for age. Between January, 1997, and January, 2012, we obtained data for our first study from 40 LRRK2 mutation carriers, 63 individuals with sporadic Parkinson's disease, and 35 healthy controls. We identified significant group differences in striatal dopamine transporter binding (all age ranges in caudate and putamen, p<0·0001) and 18 F-FDOPA uptake (in caudate: age ≤50 years, p=0·0002; all other age ranges, p<0·0001; in putamen: all age ranges, p<0·0001). LRRK2 mutation carriers with manifest Parkinson's disease (n=15) had reduced striatal dopamine transporter binding and 18 F-FDOPA uptake, comparable with amounts seen in individuals with sporadic Parkinson's disease of similar duration. LRRK2 mutation carriers without manifest Parkinson's disease (n=25) had greater 18 F-FDOPA uptake and dopamine transporter binding than did individuals with sporadic Parkinson's disease, with 18 F-FDOPA uptake comparable with controls and dopamine transporter binding lower than in controls. Between November, 2012, and May, 2016, we obtained data for our second study from 16 LRRK2 mutation carriers, 13 individuals with sporadic Parkinson's disease, and nine healthy controls. Nine LRRK2 mutation carriers without manifest Parkinson's disease had significantly elevated serotonin transporter binding in the hypothalamus (compared with controls, individuals with LRRK2 Parkinson's disease, and people with sporadic Parkinson's disease, p<0·0001), striatum (compared with people with sporadic Parkinson's disease, p=0·02), and brainstem (compared with LRRK2 mutation carriers with manifest Parkinson's disease, p=0·01), after adjustment for age. Serotonin transporter binding in the cortex did not differ significantly between groups after age adjustment. Striatal VMAT2 binding was reduced in all individuals with manifest Parkinson's disease and reduced asymmetrically in one LRRK2 mutation carrier without manifest disease. Dopaminergic and serotonergic changes progress in a similar fashion in LRRK2 mutation carriers with manifest Parkinson's disease and individuals with sporadic Parkinson's disease, but LRRK2 mutation carriers without manifest Parkinson's disease show increased serotonin transporter binding in the striatum, brainstem, and hypothalamus, possibly reflecting compensatory changes in serotonergic innervation preceding the motor onset of Parkinson's disease. Increased serotonergic innervation might contribute to clinical differences in LRRK2 Parkinson's disease, including the emergence of non-motor symptoms and, potentially, differences in the long-term response to levodopa. Canada Research Chairs, Michael J Fox Foundation, National Institutes of Health, Pacific Alzheimer Research Foundation, Pacific Parkinson's Research Institute, National Research Council of Canada. Copyright © 2017 Elsevier Ltd. All rights reserved.
-
DOE Office of Scientific and Technical Information (OSTI.GOV)
Zahn, Karl E.; Averill, April; Wallace, Susan S.
5-Hydroxycytosine (5-OHC) is a stable oxidation product of cytosine associated with an increased frequency of C {yields} T transition mutations. When this lesion escapes recognition by the base excision repair pathway and persists to serve as a templating base during DNA synthesis, replicative DNA polymerases often misincorporate dAMP at the primer terminus, which can lead to fixation of mutations and subsequent disease. To characterize the dynamics of DNA synthesis opposite 5-OHC, we initiated a comparison of unmodified dCMP to 5-OHC, 5-fluorocytosine (5-FC), and 5-methylcytosine (5-MEC) in which these bases act as templates in the active site of RB69 gp43, amore » high-fidelity DNA polymerase sharing homology with human replicative DNA polymerases. This study presents the first crystal structure of any DNA polymerase binding this physiologically important premutagenic DNA lesion, showing that while dGMP is stabilized by 5-OHC through normal Watson-Crick base pairing, incorporation of dAMP leads to unstacking and instability in the template. Furthermore, the electronegativity of the C5 substituent appears to be important in the miscoding potential of these cytosine-like templates. While dAMP is incorporated opposite 5-OHC {approx}5 times more efficiently than opposite unmodified dCMP, an elevated level of incorporation is also observed opposite 5-FC but not 5-MEC. Taken together, these data imply that the nonuniform templating by 5-OHC is due to weakened stacking capabilities, which allows dAMP incorporation to proceed in a manner similar to that observed opposite abasic sites.« less
-
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.
-
Liu, Sheng-Jie; Wang, Jiang-Yi; Peng, Shuang-He; Li, Teng; Ning, Xiang-Hui; Hong, Bao-An; Liu, Jia-Yuan; Wu, Peng-Jie; Zhou, Bo-Wen; Zhou, Jing-Cheng; Qi, Nie-Nie; Peng, Xiang; Zhang, Jiu-Feng; Ma, Kai-Fang; Cai, Lin; Gong, Kan
2018-03-29
PurposeVon Hippel-Lindau (VHL) disease is a rare hereditary cancer syndrome that reduces life expectancy. We aimed to construct a more valuable genotype-phenotype correlation based on alterations in VHL protein (pVHL).MethodsVHL patients (n = 339) were recruited and grouped based on mutation types: HIF-α binding site missense (HM) mutations, non-HIF-α binding site missense (nHM) mutations, and truncating (TR) mutations. Age-related risks of VHL-associated tumors and patient survival were compared.ResultsMissense mutations conferred an increased risk of pheochromocytoma (HR = 1.854, p = 0.047) compared with truncating mutations. The risk of pheochromocytoma was lower in the HM group than in the nHM group (HR = 0.298, p = 0.003) but was similar between HM and TR groups (HR = 0.901, p = 0.810). Patients in the nHM group had a higher risk of pheochromocytoma (HR = 3.447, p < 0.001) and lower risks of central nervous system hemangioblastoma (CHB) (HR = 0.700, p = 0.045), renal cell carcinoma (HR = 0.610, p = 0.024), and pancreatic tumor (HR = 0.382, p < 0.001) than those in the combined HM and TR (HMTR) group. Moreover, nHM mutations were independently associated with better overall survival (HR = 0.345, p = 0.005) and CHB-specific survival (HR = 0.129, p = 0.005) than HMTR mutations.ConclusionThe modified genotype-phenotype correlation links VHL gene mutation, substrate binding site, and phenotypic diversity (penetrance and survival), and provides more accurate information for genetic counseling and pathogenesis studies.Genetics in Medicine advance online publication, 29 March 2018; doi:10.1038/gim.2017.261.
-
Molecular basis for the Kallmann syndrome-linked fibroblast growth factor receptor mutation
DOE Office of Scientific and Technical Information (OSTI.GOV)
Thurman, Ryan D.; Kathir, Karuppanan Muthusamy; Rajalingam, Dakshinamurthy
Highlights: Black-Right-Pointing-Pointer The structural basis of the Kallmann syndrome is elucidated. Black-Right-Pointing-Pointer Kallmann syndrome mutation (A168S) induces a subtle conformational change(s). Black-Right-Pointing-Pointer Structural interactions mediated by beta-sheet G are most perturbed. Black-Right-Pointing-Pointer Ligand (FGF)-receptor interaction(s) is completely abolished by Kallmann mutation. Black-Right-Pointing-Pointer Kallmann mutation directly affects the FGF signaling process. -- Abstract: Kallmann syndrome (KS) is a developmental disease that expresses in patients as hypogonadotropic hypogonadism and anosmia. KS is commonly associated with mutations in the extracellular D2 domain of the fibroblast growth factor receptor (FGFR). In this study, for the first time, the molecular basis for the FGFR associatedmore » KS mutation (A168S) is elucidated using a variety of biophysical experiments, including multidimensional NMR spectroscopy. Secondary and tertiary structural analysis using far UV circular dichroism, fluorescence and limited trypsin digestion assays suggest that the KS mutation induces subtle tertiary structure change in the D2 domain of FGFR. Results of isothermal titration calorimetry experiments show the KS mutation causes a 10-fold decrease in heparin binding affinity and also a complete loss in ligand (FGF-1) binding. {sup 1}H-{sup 15}N chemical perturbation data suggest that complete loss in the ligand (FGF) binding affinity is triggered by a subtle conformational change that disrupts crucial structural interactions in both the heparin and the FGF binding sites in the D2 domain of FGFR. The novel findings reported in this study are expected to provide valuable clues toward a complete understanding of the other genetic diseases linked to mutations in the FGFR.« less
-
Dobrowolski, Steven F; McKinney, Jason T; Amat di San Filippo, Cristina; Giak Sim, Keow; Wilcken, Bridget; Longo, Nicola
2005-03-01
Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation resulting from defective carnitine transport. This disease is caused by mutations in the OCTN2 carnitine transporter encoded by the SLC22A5 gene. Here we validate dye-binding/high-resolution thermal denaturation as a screening procedure to identify novel mutations in this gene. This procedure is based on the amplification of DNA by PCR in capillaries with the dsDNA binding dye LCGreen I. The PCR reaction is then analyzed in the same capillary by high-resolution thermal denaturation. Samples with abnormal melting profiles are sequenced. This technique correctly identified all known patients who were compound heterozygotes for different mutations in the carnitine transporter gene and about 30% of homozygous patients. The remaining 70% of homozygous patients were identified by a second amplification, in which the patient's DNA was mixed with the DNA of a normal control. This screening system correctly identified eight novel mutations and both abnormal alleles in six new families with primary carnitine deficiency. The causative role of the missense mutations identified (c.3G>T/p.M1I, c.695C>T/p.T232M, and c.1403 C>G/p.T468R) was confirmed by expression in Chinese hamster ovary (CHO) cells. These results expand the mutational spectrum in primary carnitine deficiency and indicate dye-binding/high-resolution thermal denaturation as an ideal system to screen for mutations in diseases with no prevalent molecular alteration. (c) 2005 Wiley-Liss, Inc.
-
NASA Technical Reports Server (NTRS)
Setlik, R. F.; Meyer, D. J.; Shibata, M.; Roskwitalski, R.; Ornstein, R. L.; Rein, R.
1994-01-01
We present a full-coordinate model of residues 1-319 of the polymerase domain of HIV-I reverse transcriptase. This model was constructed from the x-ray crystallographic structure of Jacobo-Molina et al. (Jacobo-Molina et al., P.N.A.S. USA 90, 6320-6324 (1993)) which is currently available to the degree of C-coordinates. The backbone and side-chain atoms were constructed using the MAXSPROUT suite of programs (L. Holm and C. Sander, J. Mol. Biol. 218, 183-194 (1991)) and refined through molecular modeling. A seven base pair A-form dsDNA was positioned in the nucleic acid binding cleft to represent the template-primer complex. The orientation of the template-primer complex in the nucleic acid binding cleft was guided by the positions of phosphorus atoms in the crystal structure.
-
Schneider, Katja U; Marchini, Antonio; Sabherwal, Nitin; Röth, Ralph; Niesler, Beate; Marttila, Tiina; Blaschke, Rüdiger J; Lawson, Margaret; Dumic, Miroslav; Rappold, Gudrun
2005-07-01
Haploinsufficiency of the short stature homeobox gene SHOX has been found in patients with idiopathic short stature (ISS) and Leri-Weill dyschondrosteosis (LWD). In addition to complete gene deletions and nonsense mutations, several missense mutations have been identified in both patient groups, leading to amino acid substitutions in the SHOX protein. The majority of missense mutations were found to accumulate in the region encoding the highly conserved homeodomain of the paired-like type. In this report, we investigated nine different amino acid exchanges in the homeodomain of SHOX patients with ISS and LWD. We were able show that these mutations cause an alteration of the biological function of SHOX by loss of DNA binding, reduced dimerization ability, and/or impaired nuclear translocation. Additionally, one of the mutations (c.458G>T, p.R153L) is defective in transcriptional activation even though it is still able to bind to DNA, dimerize, and translocate to the nucleus. Thus, we demonstrate that single missense mutations in the homeodomain fundamentally impair SHOX key functions, thereby leading to the phenotype observed in patients with LWD and ISS.
-
A microsphere-based assay for mutation analysis of the biotinidase gene using dried blood spots
Lindau-Shepard, Barbara; Janik, David K.; Pass, Kenneth A.
2012-01-01
Biotinidase deficiency is an autosomal recessive syndrome caused by defects in the biotinidase gene, the product of which affects biotin metabolism. Newborn screening (NBS) for biotinidase deficiency can identify affected infants prior to onset of symptoms; biotin supplementation can resolve or prevent the clinical features. In NBS, dry blood spots (DBS) are usually tested for biotinidase enzyme activity by colorimetric analysis. By taking advantage of the multiplexing capabilities of the Luminex platform, we have developed a microsphere-based array genotyping method for the simultaneous detection of six disease causing mutations in the biotinidase gene, thereby permitting a second tier of molecular analysis. Genomic DNA was extracted from 3.2 mm DBS. Biotinidase gene sequences, containing the mutations of interest, were amplified by multiplexed polymerase chain reaction, followed by multiplexed allele-specific primer extension using universally tagged genotyping primers. The products were then hybridized to anti-tag carrying xTAG microspheres and detected on the Luminex platform. Genotypes were verified by sequencing. Genotyping results of 22 known biotinidase deficient samples by our xTAG biotinidase assay was in concordance with the results obtained from DNA sequencing, for all 6 mutations used in our panel. These results indicate that genotyping by an xTAG microsphere-based array is accurate, flexible, and can be adapted for high-throughput. Since NBS for biotinidase deficiency is by enzymatic assay, less than optimal quality of the DBS itself can compromise enzyme activity, while the DNA from these samples mostly remains unaffected. This assay warrants evaluation as a viable complement to the biotinidase semi-quantitative colorimetric assay. PMID:27625817
-
Adachi, Kaori
2014-03-01
At the Division of Functional Genomics, Research Center for Bioscience and Technology, Tottori University, we have been making an effort to establish a genetic testing facility that can provide the same screening procedures conducted worldwide. Direct Sequencing of PCR products is the main method to detect point mutations, small deletions and insertions. Multiplex Ligation-dependent Probe Amplification (MLPA) was used to detect large deletions or insertions. Expansion of the repeat was analyzed for triplet repeat diseases. Original primers were constructed for 41 diseases when the reported primers failed to amplify the gene. Prediction of functional effects of human nsSNPs (PolyPhen) was used for evaluation of novel mutations. From January 2000 to September 2013, a total of 1,006 DNA samples were subjected to genetic testing in the Division of Functional Genomics, Research Center for Bioscience and Technology, Tottori University. The hospitals that requested genetic testing were located in 43 prefectures in Japan and in 11 foreign countries. The genetic testing covered 62 diseases, and mutations were detected in 287 out of 1,006 with an average mutation detection rate of 24.7%. There were 77 samples for prenatal diagnosis. The number of samples has rapidly increased since 2010. In 2013, the next-generation sequencers were introduced in our facility and are expected to provide more comprehensive genetic testing in the near future. Nowadays, genetic testing is a popular and powerful tool for diagnosis of many genetic diseases. Our genetic testing should be further expanded in the future.
-
Hassan, Mubashir; Abbas, Qamar; Raza, Hussain; Moustafa, Ahmed A; Seo, Sung-Yum
2017-07-25
Misfolding and structural alteration in proteins lead to serious malfunctions and cause various diseases in humans. Mutations at the active binding site in tyrosinase impair structural stability and cause lethal albinism by abolishing copper binding. To evaluate the histidine mutational effect, all mutated structures were built using homology modelling. The protein sequence was retrieved from the UniProt database, and 3D models of original and mutated human tyrosinase sequences were predicted by changing the residual positions within the target sequence separately. Structural and mutational analyses were performed to interpret the significance of mutated residues (N 180 , R 202 , Q 202 , R 211 , Y 363 , R 367 , Y 367 and D 390 ) at the active binding site of tyrosinases. CSpritz analysis depicted that 23.25% residues actively participate in the instability of tyrosinase. The accuracy of predicted models was confirmed through online servers ProSA-web, ERRAT score and VERIFY 3D values. The theoretical pI and GRAVY generated results also showed the accuracy of the predicted models. The CCA negative correlation results depicted that the replacement of mutated residues at His within the active binding site disturbs the structural stability of tyrosinases. The predicted CCA scores of Tyr 367 (-0.079) and Q/R 202 (0.032) revealed that both mutations have more potential to disturb the structural stability. MD simulation analyses of all predicted models justified that Gln 202 , Arg 202 , Tyr 367 and D 390 replacement made the protein structures more susceptible to destabilization. Mutational results showed that the replacement of His with Q/R 202 and Y/R 363 has a lethal effect and may cause melanin associated diseases such as OCA1. Taken together, our computational analysis depicts that the mutated residues such as Q/R 202 and Y/R 363 actively participate in instability and misfolding of tyrosinases, which may govern OCA1 through disturbing the melanin biosynthetic pathway.
-
ATP interacts with the CPVT mutation-associated central domain of the cardiac ryanodine receptor.
Blayney, Lynda; Beck, Konrad; MacDonald, Ewan; D'Cruz, Leon; Nomikos, Michail; Griffiths, Julia; Thanassoulas, Angelos; Nounesis, George; Lai, F Anthony
2013-10-01
This study was designed to determine whether the cardiac ryanodine receptor (RyR2) central domain, a region associated with catecholamine polymorphic ventricular tachycardia (CPVT) mutations, interacts with the RyR2 regulators, ATP and the FK506-binding protein 12.6 (FKBP12.6). Wild-type (WT) RyR2 central domain constructs (G(2236)to G(2491)) and those containing the CPVT mutations P2328S and N2386I, were expressed as recombinant proteins. Folding and stability of the proteins were examined by circular dichroism (CD) spectroscopy and guanidine hydrochloride chemical denaturation. The far-UV CD spectra showed a soluble stably-folded protein with WT and mutant proteins exhibiting a similar secondary structure. Chemical denaturation analysis also confirmed a stable protein for both WT and mutant constructs with similar two-state unfolding. ATP and caffeine binding was measured by fluorescence spectroscopy. Both ATP and caffeine bound with an EC50 of ~200-400μM, and the affinity was the same for WT and mutant constructs. Sequence alignment with other ATP binding proteins indicated the RyR2 central domain contains the signature of an ATP binding pocket. Interaction of the central domain with FKBP12.6 was tested by glutaraldehyde cross-linking and no association was found. The RyR2 central domain, expressed as a 'correctly' folded recombinant protein, bound ATP in accord with bioinformatics evidence of conserved ATP binding sequence motifs. An interaction with FKBP12.6 was not evident. CPVT mutations did not disrupt the secondary structure nor binding to ATP. Part of the RyR2 central domain CPVT mutation cluster, can be expressed independently with retention of ATP binding. Copyright © 2013 Elsevier B.V. All rights reserved.
-
Kowalsky, Caitlin A; Whitehead, Timothy A
2016-12-01
The comprehensive sequence determinants of binding affinity for type I cohesin toward dockerin from Clostridium thermocellum and Clostridium cellulolyticum was evaluated using deep mutational scanning coupled to yeast surface display. We measured the relative binding affinity to dockerin for 2970 and 2778 single point mutants of C. thermocellum and C. cellulolyticum, respectively, representing over 96% of all possible single point mutants. The interface ΔΔG for each variant was reconstructed from sequencing counts and compared with the three independent experimental methods. This reconstruction results in a narrow dynamic range of -0.8-0.5 kcal/mol. The computational software packages FoldX and Rosetta were used to predict mutations that disrupt binding by more than 0.4 kcal/mol. The area under the curve of receiver operator curves was 0.82 for FoldX and 0.77 for Rosetta, showing reasonable agreements between predictions and experimental results. Destabilizing mutations to core and rim positions were predicted with higher accuracy than support positions. This benchmark dataset may be useful for developing new computational prediction tools for the prediction of the mutational effect on binding affinities for protein-protein interactions. Experimental considerations to improve precision and range of the reconstruction method are discussed. Proteins 2016; 84:1914-1928. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.
-
DOE Office of Scientific and Technical Information (OSTI.GOV)
Ti, Shih-Chieh; Pamula, Melissa C.; Howes, Stuart C.
The assembly of microtubule-based cellular structures depends on regulated tubulin polymerization and directional transport. In this research, we have purified and characterized tubulin heterodimers that have human β-tubulin isotype III (TUBB3), as well as heterodimers with one of two β-tubulin mutations (D417H or R262H). Both point mutations are proximal to the kinesin-binding site and have been linked to an ocular motility disorder in humans. Compared to wild-type, microtubules with these mutations have decreased catastrophe frequencies and increased average lifetimes of plus- and minus-end-stabilizing caps. Importantly, the D417H mutation does not alter microtubule lattice structure or Mal3 binding to growing filaments.more » Instead, this mutation reduces the affinity of tubulin for TOG domains and colchicine, suggesting that the distribution of tubulin heterodimer conformations is changed. Together, our findings reveal how residues on the surface of microtubules, distal from the GTP-hydrolysis site and inter-subunit contacts, can alter polymerization dynamics at the plus- and minus-ends of microtubules.« less
-
Mori, Yoshikazu; Ogawa, Kazuo; Warabi, Eiji; Yamamoto, Masahiro; Hirokawa, Takatsugu
2016-01-01
Transient receptor potential vanilloid type 1 (TRPV1) is a non-selective cation channel and a multimodal sensor protein. Since the precise structure of TRPV1 was obtained by electron cryo-microscopy, the binding mode of representative agonists such as capsaicin and resiniferatoxin (RTX) has been extensively characterized; however, detailed information on the binding mode of other vanilloids remains lacking. In this study, mutational analysis of human TRPV1 was performed, and four agonists (capsaicin, RTX, [6]-shogaol and [6]-gingerol) were used to identify amino acid residues involved in ligand binding and/or modulation of proton sensitivity. The detailed binding mode of each ligand was then simulated by computational analysis. As a result, three amino acids (L518, F591 and L670) were newly identified as being involved in ligand binding and/or modulation of proton sensitivity. In addition, in silico docking simulation and a subsequent mutational study suggested that [6]-gingerol might bind to and activate TRPV1 in a unique manner. These results provide novel insights into the binding mode of various vanilloids to the channel and will be helpful in developing a TRPV1 modulator. PMID:27606946
-
Sartorato, Paola; Lapeyraque, Anne-Laure; Armanini, Decio; Kuhnle, Ursula; Khaldi, Yasmina; Salomon, Rémi; Abadie, Véronique; Di Battista, Eliana; Naselli, Arturo; Racine, Alain; Bosio, Maurizio; Caprio, Massimiliano; Poulet-Young, Véronique; Chabrolle, Jean-Pierre; Niaudet, Patrick; De Gennes, Christiane; Lecornec, Marie-Hélène; Poisson, Elodie; Fusco, Anna Maria; Loli, Paola; Lombès, Marc; Zennaro, Maria-Christina
2003-06-01
We have analyzed the human mineralocorticoid receptor (hMR) gene in 14 families with autosomal dominant or sporadic pseudohypoaldosteronism (PHA1), a rare form of mineralocorticoid resistance characterized by neonatal renal salt wasting and failure to thrive. Six heterozygous mutations were detected. Two frameshift mutations in exon 2 (insT1354, del8bp537) and one nonsense mutation in exon 4 (C2157A, Cys645stop) generate truncated proteins due to premature stop codons. Three missense mutations (G633R, Q776R, L979P) differently affect hMR function. The DNA binding domain mutant R633 exhibits reduced maximal transactivation, although its binding characteristics and ED(50) of transactivation are comparable with wild-type hMR. Ligand binding domain mutants R776 and P979 present reduced or absent aldosterone binding, respectively, which is associated with reduced or absent ligand-dependent transactivation capacity. Finally, P979 possesses a transdominant negative effect on wild-type hMR activity, whereas mutations G633R and Q776R probably result in haploinsufficiency in PHA1 patients. We conclude that hMR mutations are a common feature of autosomal dominant PHA1, being found in 70% of our familial cases. Their absence in some families underscores the importance of an extensive investigation of the hMR gene and the role of precise diagnostic procedures to allow for identification of other genes potentially involved in the disease.
-
Cini, Giulia; Mezzavilla, Massimo; Della Puppa, Lara; Cupelli, Elisa; Fornasin, Alessio; D'Elia, Angela Valentina; Dolcetti, Riccardo; Damante, Giuseppe; Bertok, Sara; Miolo, Gianmaria; Maestro, Roberta; de Paoli, Paolo; Amoroso, Antonio; Viel, Alessandra
2016-02-06
About 20 % of hereditary breast cancers are caused by mutations in BRCA1 and BRCA2 genes. Since BRCA1 and BRCA2 mutations may be spread throughout the gene, genetic testing is usually performed by direct sequencing of entire coding regions. In some populations, especially if relatively isolated, a few number of recurrent mutations is reported, sometimes caused by founder effect. BRCA1 and BRCA2 screening for mutations was carried out on 1114 breast and/or ovarian cancer patients complying with the eligibility criteria for BRCA testing. Haplotype analysis was performed on the probands carrying recurrent mutations and their relatives, using two sets of microsatellite markers covering the BRCA1 (D17S588, D17S806, D17S902, D17S1325, D17S855, D17S1328, D17S800, and D17S250) and BRCA2 (D13S220, D13S267, D13S171, D13S1701, D13S1698, D13S260, D13S290, D13S1246) loci. The DMLE + 2.2 software was used to estimate the age of BRCA1 c.676delT and BRCA2 c.7806-2A > G. A multiplex PCR and two different primer extension assays were optimized and used for genotyping the recurrent mutations of the two genes. In the time frame of almost 20 years of genetic testing, we have found that five BRCA1 and three BRCA2 mutations are recurrent in a substantial subset of carriers from North-East Italy and neighboring Istria, where they represent more than 50 % of all mutations. Microsatellite analyses identified a common haplotype of different length for each mutation. Age estimation of BRCA1 c.676delT and BRCA2 c.7806-2A > G mutations revealed that they arose in the Friuli Venezia Giulia area about 86 and 94 generations ago, respectively. Suggestion of an association between BRCA2 c.7806-2A > G and risk of breast cancer in males has emerged. Finally, we developed a simple and efficient pre-screening test, performing an in-house primer extension SNaPshot® assay for the rapid identification of the eight recurrent mutations. Proofs of common ancestry has been obtained for the eight recurrent mutations. The observed genotype-phenotype correlation and the proposed rapid mutation detection strategy could improve the clinical management of breast and ovarian patients in North-East of Italy and neighboring geographic areas.
-
Suetomi, Takeshi; Yano, Masafumi; Uchinoumi, Hitoshi; Fukuda, Masakazu; Hino, Akihiro; Ono, Makoto; Xu, Xiaojuan; Tateishi, Hiroki; Okuda, Shinichi; Doi, Masahiro; Kobayashi, Shigeki; Ikeda, Yasuhiho; Yamamoto, Takeshi; Ikemoto, Noriaki; Matsuzaki, Masunori
2011-01-01
Background The molecular mechanism by which catecholaminergic polymorphic ventricular tachycardia (CPVT) is induced by single amino acid mutations within the cardiac ryanodine receptor (RyR2) remains elusive. Here, we investigated mutation-induced conformational defects of RyR2 using a knock-in (KI) mouse model expressing the human CPVT-associated RyR2 mutant (S2246L; Serine to Leucine mutation at the residue 2246). Methods and Results All KI mice we examined produced VT after exercise on a treadmill. cAMP-dependent increase in the frequency of Ca2+ sparks was more pronounced in saponin-permeabilized KI cardiomyocytes than in WT cardiomyocytes. Site-directed fluorescent labeling and quartz microbalance assays of the specific binding of DP2246 (a peptide corresponding to the 2232–2266 region: the 2246 domain) showed that DP2246 binds with the K201-binding sequence of RyR2 (1741– 2270). Introduction of S2246L mutation into the DP2246 increased the affinity of peptide binding. Fluorescence quench assays of inter-domain interactions within RyR2 showed that tight interaction of the 2246 domain/K201-binding domain is coupled with domain unzipping of the N-terminal (1-600)/central (2000–2500) domain pair in an allosteric manner. Dantrolene corrected the mutation-caused domain unzipping of the domain switch, and stopped the exercise-induced ventricular tachycardia. Conclusions The CPVT-linked mutation of RyR2, S2246L, causes an abnormally tight local sub-domain/sub-domain interaction within the central domain involving the mutation site, which induces defective interaction between the N-terminal and central domains. This results in an erroneous activation of Ca2+ channel in a diastolic state reflecting on the increased Ca2+ spark frequency, which then leads to lethal arrhythmia. PMID:21768539
-
Mazhab-Jafari, Mohammad T; Marshall, Christopher B; Smith, Matthew J; Gasmi-Seabrook, Geneviève M C; Stathopulos, Peter B; Inagaki, Fuyuhiko; Kay, Lewis E; Neel, Benjamin G; Ikura, Mitsuhiko
2015-05-26
K-RAS4B (Kirsten rat sarcoma viral oncogene homolog 4B) is a prenylated, membrane-associated GTPase protein that is a critical switch for the propagation of growth factor signaling pathways to diverse effector proteins, including rapidly accelerated fibrosarcoma (RAF) kinases and RAS-related protein guanine nucleotide dissociation stimulator (RALGDS) proteins. Gain-of-function KRAS mutations occur frequently in human cancers and predict poor clinical outcome, whereas germ-line mutations are associated with developmental syndromes. However, it is not known how these mutations affect K-RAS association with biological membranes or whether this impacts signal transduction. Here, we used solution NMR studies of K-RAS4B tethered to nanodiscs to investigate lipid bilayer-anchored K-RAS4B and its interactions with effector protein RAS-binding domains (RBDs). Unexpectedly, we found that the effector-binding region of activated K-RAS4B is occluded by interaction with the membrane in one of the NMR-observable, and thus highly populated, conformational states. Binding of the RAF isoform ARAF and RALGDS RBDs induced marked reorientation of K-RAS4B from the occluded state to RBD-specific effector-bound states. Importantly, we found that two Noonan syndrome-associated mutations, K5N and D153V, which do not affect the GTPase cycle, relieve the occluded orientation by directly altering the electrostatics of two membrane interaction surfaces. Similarly, the most frequent KRAS oncogenic mutation G12D also drives K-RAS4B toward an exposed configuration. Further, the D153V and G12D mutations increase the rate of association of ARAF-RBD with lipid bilayer-tethered K-RAS4B. We revealed a mechanism of K-RAS4B autoinhibition by membrane sequestration of its effector-binding site, which can be disrupted by disease-associated mutations. Stabilizing the autoinhibitory interactions between K-RAS4B and the membrane could be an attractive target for anticancer drug discovery.
-
Suetomi, Takeshi; Yano, Masafumi; Uchinoumi, Hitoshi; Fukuda, Masakazu; Hino, Akihiro; Ono, Makoto; Xu, Xiaojuan; Tateishi, Hiroki; Okuda, Shinichi; Doi, Masahiro; Kobayashi, Shigeki; Ikeda, Yasuhiro; Yamamoto, Takeshi; Ikemoto, Noriaki; Matsuzaki, Masunori
2011-08-09
The molecular mechanism by which catecholaminergic polymorphic ventricular tachycardia is induced by single amino acid mutations within the cardiac ryanodine receptor (RyR2) remains elusive. In the present study, we investigated mutation-induced conformational defects of RyR2 using a knockin mouse model expressing the human catecholaminergic polymorphic ventricular tachycardia-associated RyR2 mutant (S2246L; serine to leucine mutation at the residue 2246). All knockin mice we examined produced ventricular tachycardia after exercise on a treadmill. cAMP-dependent increase in the frequency of Ca²⁺ sparks was more pronounced in saponin-permeabilized knockin cardiomyocytes than in wild-type cardiomyocytes. Site-directed fluorescent labeling and quartz microbalance assays of the specific binding of DP2246 (a peptide corresponding to the 2232 to 2266 region: the 2246 domain) showed that DP2246 binds with the K201-binding sequence of RyR2 (1741 to 2270). Introduction of S2246L mutation into the DP2246 increased the affinity of peptide binding. Fluorescence quench assays of interdomain interactions within RyR2 showed that tight interaction of the 2246 domain/K201-binding domain is coupled with domain unzipping of the N-terminal (1 to 600)/central (2000 to 2500) domain pair in an allosteric manner. Dantrolene corrected the mutation-caused domain unzipping of the domain switch and stopped the exercise-induced ventricular tachycardia. The catecholaminergic polymorphic ventricular tachycardia-linked mutation of RyR2, S2246L, causes an abnormally tight local subdomain-subdomain interaction within the central domain involving the mutation site, which induces defective interaction between the N-terminal and central domains. This results in an erroneous activation of Ca²⁺ channel in a diastolic state reflecting on the increased Ca²⁺ spark frequency, which then leads to lethal arrhythmia.
-
Takemura, Kazuhiro; Hanawa-Suetsugu, Kyoko; Suetsugu, Shiro; Kitao, Akio
2017-07-28
The BAR domain superfamily proteins sense or induce curvature in membranes. The inverse-BAR domain (I-BAR) is a BAR domain that forms a straight "zeppelin-shaped" dimer. The mechanisms by which IRSp53 I-BAR binds to and deforms a lipid membrane are investigated here by all-atom molecular dynamics simulation (MD), binding energy analysis, and the effects of mutation experiments on filopodia on HeLa cells. I-BAR adopts a curved structure when crystallized, but adopts a flatter shape in MD. The binding of I-BAR to membrane was stabilized by ~30 salt bridges, consistent with experiments showing that point mutations of the interface residues have little effect on the binding affinity whereas multiple mutations have considerable effect. Salt bridge formation increases the local density of lipids and deforms the membrane into a concave shape. In addition, the point mutations that break key intra-molecular salt bridges within I-BAR reduce the binding affinity; this was confirmed by expressing these mutants in HeLa cells and observing their effects. The results indicate that the stiffness of I-BAR is important for membrane deformation, although I-BAR does not act as a completely rigid template.
-
Anandapadamanaban, Madhanagopal; Pilstål, Robert; Andresen, Cecilia; Trewhella, Jill; Moche, Martin; Wallner, Björn; Sunnerhagen, Maria
2016-08-02
MexR is a repressor of the MexAB-OprM multidrug efflux pump operon of Pseudomonas aeruginosa, where DNA-binding impairing mutations lead to multidrug resistance (MDR). Surprisingly, the crystal structure of an MDR-conferring MexR mutant R21W (2.19 Å) presented here is closely similar to wild-type MexR. However, our extended analysis, by molecular dynamics and small-angle X-ray scattering, reveals that the mutation stabilizes a ground state that is deficient of DNA binding and is shared by both mutant and wild-type MexR, whereas the DNA-binding state is only transiently reached by the more flexible wild-type MexR. This population shift in the conformational ensemble is effected by mutation-induced allosteric coupling of contact networks that are independent in the wild-type protein. We propose that the MexR-R21W mutant mimics derepression by small-molecule binding to MarR proteins, and that the described allosteric model based on population shifts may also apply to other MarR family members. Copyright © 2016 Elsevier Ltd. All rights reserved.
-
Lukman, Suryani; Lane, David P.; Verma, Chandra S.
2013-01-01
The transcription factor p53 regulates cellular integrity in response to stress. p53 is mutated in more than half of cancerous cells, with a majority of the mutations localized to the DNA binding domain (DBD). In order to map the structural and dynamical features of the DBD, we carried out multiple copy molecular dynamics simulations (totaling 0.8 μs). Simulations show the loop 1 to be the most dynamic element among the DNA-contacting loops (loops 1-3). Loop 1 occupies two major conformational states: extended and recessed; the former but not the latter displays correlations in atomic fluctuations with those of loop 2 (~24 Å apart). Since loop 1 binds to the major groove whereas loop 2 binds to the minor groove of DNA, our results begin to provide some insight into the possible mechanism underpinning the cooperative nature of DBD binding to DNA. We propose (1) a novel mechanism underlying the dynamics of loop 1 and the possible tread-milling of p53 on DNA and (2) possible mutations on loop 1 residues to restore the transcriptional activity of an oncogenic mutation at a distant site. PMID:24324553
-
The molecular genetic analysis of the expanding pachyonychia congenita case collection
Wilson, NJ; O'Toole, EA; Milstone, LM; Hansen, CD; Shepherd, AA; Al-Asadi, E; Schwartz, ME; McLean, WHI; Sprecher, E; Smith, FJD
2014-01-01
Background Pachyonychia congenita (PC) is a rare autosomal dominant keratinizing disorder characterized by severe, painful, palmoplantar keratoderma and nail dystrophy, often accompanied by oral leucokeratosis, cysts and follicular keratosis. It is caused by mutations in one of five keratin genes: KRT6A, KRT6B, KRT6C, KRT16 or KRT17. Objectives To identify mutations in 84 new families with a clinical diagnosis of PC, recruited by the International Pachyonychia Congenita Research Registry during the last few years. Methods Genomic DNA isolated from saliva or peripheral blood leucocytes was amplified using primers specific for the PC-associated keratin genes and polymerase chain reaction products were directly sequenced. Results Mutations were identified in 84 families in the PC-associated keratin genes, comprising 46 distinct keratin mutations. Fourteen were previously unreported mutations, bringing the total number of different keratin mutations associated with PC to 105. Conclusions By identifying mutations in KRT6A, KRT6B, KRT6C, KRT16 or KRT17, this study has confirmed, at the molecular level, the clinical diagnosis of PC in these families. PMID:24611874
-
Discrimination of SHV β-Lactamase Genes by Restriction Site Insertion-PCR
Chanawong, Aroonwadee; M'Zali, Fatima Hannachi; Heritage, John; Lulitanond, Aroonlug; Hawkey, Peter Michael
2001-01-01
Restriction site insertion-PCR (RSI-PCR) is a simple, rapid technique for detection of point mutations. This technique exploits primers with one to three base mismatches near the 3′ end to modulate a restriction site. We have developed this technique to identify described mutations of the blaSHV genes for differentiation of SHV variants that cannot be distinguished easily by other techniques. To validate this method, eight standard strains were used, each producing a different SHV β-lactamase: SHV-1, SHV-2, SHV-3, SHV-4, SHV-5, SHV-6, SHV-8, and SHV-18. Mismatch primers were designed to detect mutations affecting amino acids at positions 8 (SspI), 179 (HinfI), 205 (PstI), 238 (Gly→Ala) (BsrI), and 240 (NruI) of blaSHV genes. All amplimers of the blaSHV genes used in this study yielded the predicted restriction endonuclease digestion products. In addition, this study also makes theoretical identification of blaSHV-6, blaSHV-8, and 12 novel blaSHV variants using the PCR-restriction fragment length polymorphism (RFLP) technique possible. By using a combination of PCR-RFLP and RSI-PCR techniques, up to 27 SHV variants can now be distinguished rapidly and reliably. These simple techniques are readily applied to epidemiological studies of the SHV β-lactamases and may be extended to the characterisation of other resistance determinants. PMID:11408231
-
NASA Astrophysics Data System (ADS)
Zhang, Zhe; Martiny, Virginie; Lagorce, David; Alexov, Emil; Miteva, Maria; Clemson University Team; Université Paris Diderot Team
2013-03-01
Snyder-Robinson Syndrome (SRS) is an X-linked mental retardation disorder, which is caused by defects in a particular gene coding for the spermine synthase (SMS) protein. Among the missense mutations known to be disease-causing is the G56S, which is positioned at the interface of the SMS homo-dimer. Previous computational and experimental investigations have shown that G56S mutation destabilizes the homo-dimer and thus greatly reduces the SMS enzymatic activity. In this study, we explore the possibility of mitigating the effect of G56S mutation by binding small molecules to suitable pockets around the mutation site. It is done by combined efforts of molecular dynamics simulations and in silico screening. The binding of selected molecules was calculated to fully compensate the effect of the mutation and rescue the wild type dimer affinity. This work was supported by NIH, NLM grant. No. 1R03LM009748
-
Sant'Anna, Ricardo; Almeida, Maria Rosário; Varejāo, Nathalia; Gallego, Pablo; Esperante, Sebastian; Ferreira, Priscila; Pereira-Henriques, Alda; Palhano, Fernando L; de Carvalho, Mamede; Foguel, Debora; Reverter, David; Saraiva, Maria João; Ventura, Salvador
2017-03-24
More than a hundred different Transthyretin (TTR) mutations are associated with fatal systemic amyloidoses. They destabilize the protein tetrameric structure and promote the extracellular deposition of TTR as pathological amyloid fibrils. So far, only mutations R104H and T119M have been shown to stabilize significantly TTR, acting as disease suppressors. We describe a novel A108V non-pathogenic mutation found in a Portuguese subject. This variant is more stable than wild type TTR both in vitro and in human plasma, a feature that prevents its aggregation. The crystal structure of A108V reveals that this stabilization comes from novel intra and inter subunit contacts involving the thyroxine (T 4 ) binding site. Exploiting this observation, we engineered a A108I mutation that fills the T 4 binding cavity, as evidenced in the crystal structure. This synthetic protein becomes one of the most stable TTR variants described so far, with potential application in gene and protein replacement therapies.
-
Norovirus Escape from Broadly Neutralizing Antibodies Is Limited to Allostery-Like Mechanisms
Kolawole, Abimbola O.; Smith, Hong Q.; Svoboda, Sophia A.; Lewis, Madeline S.; Sherman, Michael B.; Lynch, Gillian C.; Pettitt, B. Montgomery
2017-01-01
ABSTRACT Ideal antiviral vaccines elicit antibodies (Abs) with broad strain recognition that bind to regions that are difficult to mutate for escape. Using 10 murine norovirus (MNV) strains and 5 human norovirus (HuNoV) virus-like particles (VLPs), we identified monoclonal antibody (MAb) 2D3, which broadly neutralized all MNV strains tested. Importantly, escape mutants corresponding to this antibody were very slow to develop and were distal to those raised against our previously studied antibody, A6.2. To understand the atomic details of 2D3 neutralization, we determined the cryo-electron microscopy (cryo-EM) structure of the 2D3/MNV1 complex. Interestingly, 2D3 binds to the top of the P domain, very close to where A6.2 binds, but the only escape mutations identified to date fall well outside the contact regions of both 2D3 and A6.2. To determine how mutations in distal residues could block antibody binding, we used molecular dynamics flexible fitting simulations of the atomic structures placed into the density map to examine the 2D3/MNV1 complex and these mutations. Our findings suggest that the escape mutant, V339I, may stabilize a salt bridge network at the P-domain dimer interface that, in an allostery-like manner, affects the conformational relaxation of the P domain and the efficiency of binding. They further highlight the unusual antigenic surface bound by MAb 2D3, one which elicits cross-reactive antibodies but which the virus is unable to alter to escape neutralization. These results may be leveraged to generate norovirus (NoV) vaccines containing broadly neutralizing antibodies. IMPORTANCE The simplest and most common way for viruses to escape antibody neutralization is by mutating residues that are essential for antibody binding. Escape mutations are strongly selected for by their effect on viral fitness, which is most often related to issues of protein folding, particle assembly, and capsid function. The studies presented here demonstrated that a broadly neutralizing antibody to mouse norovirus binds to an exposed surface but that the only escape mutants that arose were distal to the antibody binding surface. To understand this finding, we performed an in silico analysis that suggested that those escape mutations blocked antibody binding by affecting structural plasticity. This kind of antigenic region—one that gives rise to broadly neutralizing antibodies but that the virus finds difficult to escape from—is therefore ideal for vaccine development. PMID:29062895
-
Ciucci, Alessandra; Palma, Carla; Manzini, Stefano; Werge, Thomas M
1998-01-01
The binding modalities of substance P and neurokinin A on the wild type and Gly166 to-Cys mutant NK1 receptors expressed on CHO cells were investigated in homologous and heterologous binding experiments using both radiolabelled substance P and neurokinin A.On the wild type NK1 receptor NKA displaces radiolabelled substance P with very low apparent affinity, despite its high-affinity binding constant (determined in homologous binding experiments). The Gly166 to-Cys substitution in the NK1 tachykinin receptor greatly enhances the apparent affinity of neurokinin A in competition for radiolabelled substance P, but it does not change the binding constant of neurokinin A. The mutation, thereby, eliminates the discrepancy between the low apparent affinity and the high binding constant of neurokinin A.On the wild type receptor the binding capacity of neurokinin A is significantly smaller than that of substance P. In contrast, the two tachykinins bind to approximately the same number of sites on the mutant receptor.Simultaneous mass action law analysis of binding data in which multiple radioligands were employed in parallel demonstrated that a one-site model was unable to accommodate all the experimental data, whereas a two-site model provided a dramatically better description.These two receptor-sites display equally high affinity for substance P, while neurokinin A strongly discriminates between a high and a low affinity component. The binding affinities of neurokinin A are not affected by the mutation, which instead specifically alters the distribution between receptor sites in favour of a high affinity neurokinin A binding form.The low apparent affinity and binding capacity of neurokinin A on the wild type receptor results from neurokinin A binding with high affinity only to a fraction of the sites labelled by substance P. The mutation increases the proportion of this site, and consequently enhances the apparent affinity and binding capacity of neurokinin A.The binding modalities of septide-like ligands (i.e. neurokinin B, SP(6-11), SP-methyl ester) are affected similarly to neurokinin A and are better resolved into two sites. The mutation leaves the affinity of these ligands for the two receptor forms unchanged, but increases the fraction of high-affinity sites. On the other hand, the binding of non-peptide and peptide antagonists (SR140.333 and FK888) behaved similarly to substance P with a single high affinity site that is unaffected by the mutation.These findings may suggest that the NK1 receptor exists in two different forms with similar affinity for substance P and NK1 antagonists, but with a high and a low affinity for neurokinin A and septide-like ligands. Hence, the Gly166 in the NK1 receptor would seem to control the distribution between a pan-reactive form and a substance P-selective form of the receptor. PMID:9786514
-
The Oncogenic Role of RhoGAPs in Basal-Like Breast Cancer
2016-04-01
somatic mutations of RhoA in peripheral T cell lymphomas (PTCLs) (16-18) and in diffuse-type gastric carcinomas (19-21). Surprisingly, unlike Rac1...Diffuse-type gastric cancers exhibited mutations in the effector binding domain of RhoA, most commonly Y42C (19-21), which prevents binding to the...Impiombato A, Perez-Garcia A, et al. Recurrent mutations in epigenetic regulators, RHOA and FYN kinase in peripheral T cell lymphomas . Nat Genet 2014;46
-
DOE Office of Scientific and Technical Information (OSTI.GOV)
Zhang, Zi Chao; Chook, Yuh Min
Mutations in the proline/tyrosine–nuclear localization signal (PY-NLS) of the Fused in Sarcoma protein (FUS) cause amyotrophic lateral sclerosis (ALS). Here we report the crystal structure of the FUS PY-NLS bound to its nuclear import receptor Karyopherinβ2 (Kapβ2; also known as Transportin). The FUS PY-NLS occupies the structurally invariant C-terminal arch of Kapβ2, tracing a path similar to that of other characterized PY-NLSs. Unlike other PY-NLSs, which generally bind Kapβ2 in fully extended conformations, the FUS peptide is atypical as its central portion forms a 2.5-turn α-helix. The Kapβ2-binding epitopes of the FUS PY-NLS consist of an N-terminal PGKM hydrophobic motif,more » a central arginine-rich α-helix, and a C-terminal PY motif. ALS mutations are found almost exclusively within these epitopes. Each ALS mutation site makes multiple contacts with Kapβ2 and mutations of these residues decrease binding affinities for Kapβ2 (K D for wild-type FUS PY-NLS is 9.5 nM) up to ninefold. Thermodynamic analyses of ALS mutations in the FUS PY-NLS show that the weakening of FUS-Kapβ2 binding affinity, the degree of cytoplasmic mislocalization, and ALS disease severity are correlated.« less
-
Liu, Meng; Song, Jinping; Shuang, Shaomin; Dong, Chuan; Brennan, John D; Li, Yingfu
2014-06-24
We report a versatile biosensing platform capable of achieving ultrasensitive detection of both small-molecule and macromolecular targets. The system features three components: reduced graphene oxide for its ability to adsorb single-stranded DNA molecules nonspecifically, DNA aptamers for their ability to bind reduced graphene oxide but undergo target-induced conformational changes that facilitate their release from the reduced graphene oxide surface, and rolling circle amplification (RCA) for its ability to amplify a primer-template recognition event into repetitive sequence units that can be easily detected. The key to the design is the tagging of a short primer to an aptamer sequence, which results in a small DNA probe that allows for both effective probe adsorption onto the reduced graphene oxide surface to mask the primer domain in the absence of the target, as well as efficient probe release in the presence of the target to make the primer available for template binding and RCA. We also made an observation that the circular template, which on its own does not cause a detectable level of probe release from the reduced graphene oxide, augments target-induced probe release. The synergistic release of DNA probes is interpreted to be a contributing factor for the high detection sensitivity. The broad utility of the platform is illustrated though engineering three different sensors that are capable of achieving ultrasensitive detection of a protein target, a DNA sequence and a small-molecule analyte. We envision that the approach described herein will find useful applications in the biological, medical, and environmental fields.
-
D'Annessa, Ilda; Gandaglia, Anna; Brivio, Elena; Stefanelli, Gilda; Frasca, Angelisa; Landsberger, Nicoletta; Di Marino, Daniele
2018-05-01
Mutations in the X-linked MECP2 gene represent the main origin of Rett syndrome, causing a profound intellectual disability in females. MeCP2 is an epigenetic transcriptional regulator containing two main functional domains: a methyl-CpG binding domain (MBD) and a transcription repression domain (TRD). Over 600 pathogenic mutations were reported to affect the whole protein; almost half of missense mutations affect the MBD. Understanding the impact of these mutations on the MBD structure and interaction with DNA will foster the comprehension of their pathogenicity and possibly genotype/phenotype correlation studies. Herein, we use molecular dynamics simulations to obtain a detailed view of the dynamics of WT and mutated MBD in the presence and absence of DNA. The pathogenic mutation Y120D is used as paradigm for our studies. Further, since the Y120 residue was previously found to be a phosphorylation site, we characterize the dynamic profile of the MBD also in the presence of Y120 phosphorylation (pY120). We found that addition of a phosphate group to Y120 or mutation in aspartic acid affect domain mobility that samples an alternative conformational space with respect to the WT, leading to impaired ability to interact with DNA. Experimental assays showing a significant reduction in the binding affinity between the mutated MBD and the DNA confirmed our predictions. Copyright © 2018 Elsevier B.V. All rights reserved.
-
Primer development to obtain complete coding sequence of HA and NA genes of influenza A/H3N2 virus.
Agustiningsih, Agustiningsih; Trimarsanto, Hidayat; Setiawaty, Vivi; Artika, I Made; Muljono, David Handojo
2016-08-30
Influenza is an acute respiratory illness and has become a serious public health problem worldwide. The need to study the HA and NA genes in influenza A virus is essential since these genes frequently undergo mutations. This study describes the development of primer sets for RT-PCR to obtain complete coding sequence of Hemagglutinin (HA) and Neuraminidase (NA) genes of influenza A/H3N2 virus from Indonesia. The primers were developed based on influenza A/H3N2 sequence worldwide from Global Initiative on Sharing All Influenza Data (GISAID) and further tested using Indonesian influenza A/H3N2 archived samples of influenza-like illness (ILI) surveillance from 2008 to 2009. An optimum RT-PCR condition was acquired for all HA and NA fragments designed to cover complete coding sequence of HA and NA genes. A total of 71 samples were successfully sequenced for complete coding sequence both of HA and NA genes out of 145 samples of influenza A/H3N2 tested. The developed primer sets were suitable for obtaining complete coding sequences of HA and NA genes of Indonesian samples from 2008 to 2009.
-
A simple PCR-based strategy for estimating species-specific contributions in chimeras and xenografts
Ealba, Erin L.; Schneider, Richard A.
2013-01-01
Many tissue-engineering approaches for repair and regeneration involve transplants between species. Yet a challenge is distinguishing donor versus host effects on gene expression. This study provides a simple molecular strategy to quantify species-specific contributions in chimeras and xenografts. Species-specific primers for reverse transcription quantitative real-time PCR (RT-qPCR) were designed by identifying silent mutations in quail, duck, chicken, mouse and human ribosomal protein L19 (RPL19). cDNA from different pairs of species was mixed in a dilution series and species-specific RPL19 primers were used to generate standard curves. Then quail cells were transplanted into transgenic-GFP chick and resulting chimeras were analyzed with species-specific primers. Fluorescence-activated cell sorting (FACS) confirmed that donor- and host-specific levels of RPL19 expression represent actual proportions of cells. To apply the RPL19 strategy, we measured Runx2 expression in quail-duck chimeras. Elevated Runx2 levels correlated with higher percentages of donor cells. Finally, RPL19 primers also discriminated mouse from human and chick. Thus, this strategy enables chimeras and/or xenografts to be screened rapidly at the molecular level. PMID:23785056
-
2014-01-01
Background The positive transcription elongation factor b (P-TEFb) plays an essential role in activating HIV genome transcription. It is recruited to the HIV LTR promoter through an interaction between the Tat viral protein and its Cyclin T1 subunit. P-TEFb activity is inhibited by direct binding of its subunit Cyclin T (1 or 2) with Hexim (1 or 2), a cellular protein, bound to the 7SK small nuclear RNA. Hexim1 competes with Tat for P-TEFb binding. Results Mutations that impair human Cyclin T1/Hexim1 interaction were searched using systematic mutagenesis of these proteins coupled with a yeast two-hybrid screen for loss of protein interaction. Evolutionary conserved Hexim1 residues belonging to an unstructured peptide located N-terminal of the dimerization domain, were found to be critical for P-TEFb binding. Random mutagenesis of the N-terminal region of Cyclin T1 provided identification of single amino-acid mutations that impair Hexim1 binding in human cells. Furthermore, conservation of critical residues supported the existence of a functional Hexim1 homologue in nematodes. Conclusions Single Cyclin T1 amino-acid mutations that impair Hexim1 binding are located on a groove between the two cyclin folds and define a surface overlapping the HIV-1 Tat protein binding surface. One residue, Y175, in the centre of this groove was identified as essential for both Hexim1 and Tat binding to P-TEFb as well as for HIV transcription. PMID:24985203
-
Wang, Jing-Fang; Chou, Kuo-Chen
2012-01-01
Human mitochondrial ornithine transporter-1 is reported in coupling with the hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome, which is a rare autosomal recessive disorder. For in-depth understanding of the molecular mechanism of the disease, it is crucially important to acquire the 3D structure of human mitochondrial ornithine transporter-1. Since no such structure is available in the current protein structure database, we have developed it via computational approaches based on the recent NMR structure of human mitochondrial uncoupling protein (Berardi MJ, Chou JJ, et al. Nature 2011, 476:109–113). Subsequently, we docked the ligand L-ornithine into the computational structure to search for the favorable binding mode. It was observed that the binding interaction for the most favorable binding mode is featured by six remarkable hydrogen bonds between the receptor and ligand, and that the most favorable binding mode shared the same ligand-binding site with most of the homologous mitochondrial carriers from different organisms, implying that the ligand-binding sites are quite conservative in the mitochondrial carriers family although their sequences similarity is very low with 20% or so. Moreover, according to our structural analysis, the relationship between the disease-causing mutations of human mitochondrial ornithine transporter-1 and the HHH syndrome can be classified into the following three categories: (i) the mutation occurs in the pseudo-repeat regions so as to change the region of the protein closer to the mitochondrial matrix; (ii) the mutation is directly affecting the substrate binding pocket so as to reduce the substrate binding affinity; (iii) the mutation is located in the structural region closer to the intermembrane space that can significantly break the salt bridge networks of the protein. These findings may provide useful insights for in-depth understanding of the molecular mechanism of the HHH syndrome and developing effective drugs against the disease. PMID:22292090
-
Schiro, Michelle M.; Stauber, Sara E.; Peterson, Tami L.; Krueger, Chateen; Darnell, Steven J.; Satyshur, Kenneth A.; Drinkwater, Norman R.; Newton, Michael A.; Hoffmann, F. Michael
2011-01-01
Background Hub proteins are connected through binding interactions to many other proteins. Smad3, a mediator of signal transduction induced by transforming growth factor beta (TGF-β), serves as a hub protein for over 50 protein-protein interactions. Different cellular responses mediated by Smad3 are the product of cell-type and context dependent Smad3-nucleated protein complexes acting in concert. Our hypothesis is that perturbation of this spectrum of protein complexes by mutation of single protein-binding hot-spots on Smad3 will have distinct consequences on Smad3-mediated responses. Methodology/Principal Findings We mutated 28 amino acids on the surface of the Smad3 MH2 domain and identified 22 Smad3 variants with reduced binding to subsets of 17 Smad3-binding proteins including Smad4, SARA, Ski, Smurf2 and SIP1. Mutations defective in binding to Smad4, e.g., D408H, or defective in nucleocytoplasmic shuttling, e.g., W406A, were compromised in modulating the expression levels of a Smad3-dependent reporter gene or six endogenous Smad3-responsive genes: Mmp9, IL11, Tnfaip6, Fermt1, Olfm2 and Wnt11. However, the Smad3 mutants Y226A, Y297A, W326A, K341A, and E267A had distinct differences on TGF-β signaling. For example, K341A and Y226A both reduced the Smad3-mediated activation of the reporter gene by ∼50% but K341A only reduced the TGF-β inducibilty of Olfm2 in contrast to Y226A which reduced the TGF-β inducibility of all six endogenous genes as severely as the W406A mutation. E267A had increased protein binding but reduced TGF-β inducibility because it caused higher basal levels of expression. Y297A had increased TGF-β inducibility because it caused lower Smad3-induced basal levels of gene expression. Conclusions/Significance Mutations in protein binding hot-spots on Smad3 reduced the binding to different subsets of interacting proteins and caused a range of quantitative changes in the expression of genes induced by Smad3. This approach should be useful for unraveling which Smad3 protein complexes are critical for specific biological responses. PMID:21949838
-
DOE Office of Scientific and Technical Information (OSTI.GOV)
Hoetzel, Isidro; Cheevers, William P.
2005-09-01
The caprine arthritis-encephalitis (CAEV) and ovine maedi-visna (MVV) viruses are resistant to antibody neutralization, a feature shared with all other lentiviruses. Whether the CAEV gp135 receptor binding site(s) (RBS) in the functional surface envelope glycoprotein (Env) is protected from antibody binding, allowing the virus to resist neutralization, is not known. Two CAEV gp135 regions were identified by extrapolating a gp135 structural model that could affect binding of antibodies to the RBS: the V1 region and a short sequence analogous in position to the human immunodeficiency virus type 1 gp120 loop B postulated to be located between two major domains ofmore » CAEV gp135. Mutation of isoleucine-166 to alanine in the putative loop B of gp135 increased the affinity of soluble gp135 for the CAEV receptor(s) and goat monoclonal antibody (Mab) F7-299 which recognizes an epitope overlapping the gp135 RBS. The I166A mutation also stabilized or exposed the F7-299 epitope in anionic detergent buffers, indicating that the I166A mutation induces conformational changes and stabilizes the RBS of soluble gp135 and enhances Mab F7-299 binding. In contrast, the affinity of a V1 deletion mutant of gp135 for the receptor and Mab F7-299 and its structural stability did not differ from that of the wild-type gp135. However, both the I166A mutation and the V1 deletion of gp135 increased cell-to-cell fusion activity and binding of Mab F7-299 to the oligomeric Env. Therefore, the CAEV gp135 RBS is protected from antibody binding by mechanisms both dependent and independent of Env oligomerization which are disrupted by the V1 deletion and the I166A mutation, respectively. In addition, we found a correlation between side-chain {beta}-branching at amino acid position 166 and binding of Mab F7-299 to oligomeric Env and cell-to-cell fusion, suggesting local secondary structure constraints in the region around isoleucine-166 as one determinant of gp135 RBS exposure and antibody binding.« less
-
Schiro, Michelle M; Stauber, Sara E; Peterson, Tami L; Krueger, Chateen; Darnell, Steven J; Satyshur, Kenneth A; Drinkwater, Norman R; Newton, Michael A; Hoffmann, F Michael
2011-01-01
Hub proteins are connected through binding interactions to many other proteins. Smad3, a mediator of signal transduction induced by transforming growth factor beta (TGF-β), serves as a hub protein for over 50 protein-protein interactions. Different cellular responses mediated by Smad3 are the product of cell-type and context dependent Smad3-nucleated protein complexes acting in concert. Our hypothesis is that perturbation of this spectrum of protein complexes by mutation of single protein-binding hot-spots on Smad3 will have distinct consequences on Smad3-mediated responses. We mutated 28 amino acids on the surface of the Smad3 MH2 domain and identified 22 Smad3 variants with reduced binding to subsets of 17 Smad3-binding proteins including Smad4, SARA, Ski, Smurf2 and SIP1. Mutations defective in binding to Smad4, e.g., D408H, or defective in nucleocytoplasmic shuttling, e.g., W406A, were compromised in modulating the expression levels of a Smad3-dependent reporter gene or six endogenous Smad3-responsive genes: Mmp9, IL11, Tnfaip6, Fermt1, Olfm2 and Wnt11. However, the Smad3 mutants Y226A, Y297A, W326A, K341A, and E267A had distinct differences on TGF-β signaling. For example, K341A and Y226A both reduced the Smad3-mediated activation of the reporter gene by ∼50% but K341A only reduced the TGF-β inducibilty of Olfm2 in contrast to Y226A which reduced the TGF-β inducibility of all six endogenous genes as severely as the W406A mutation. E267A had increased protein binding but reduced TGF-β inducibility because it caused higher basal levels of expression. Y297A had increased TGF-β inducibility because it caused lower Smad3-induced basal levels of gene expression. Mutations in protein binding hot-spots on Smad3 reduced the binding to different subsets of interacting proteins and caused a range of quantitative changes in the expression of genes induced by Smad3. This approach should be useful for unraveling which Smad3 protein complexes are critical for specific biological responses.
-
Hb taradale [beta82(EF6)Lys-->Arg]: a novel mutation at a 2,3-diphosphoglycerate binding site.
Brennan, Stephen O; Sheen, Campbell; Chan, Tim; George, Peter M
2005-01-01
Hb Taradale [beta82(EF6)Lys-->Arg] was initially detected as a split Hb A0 peak on Hb A1c, monitoring. Red cell parameters, hemoglobin (Hb) electrophoresis and stability tests were normal. Mass spectrometry (ms) clearly identified a variant beta chain with a mass increase of 28 Da and peptide mapping located the mutation site to peptide betaT-9. DNA sequencing confirmed the presence of a novel beta82(EF6)Lys-->Arg mutation. This conservative substitution at a 2,3-diphosphoglycerate (2,3-DPG) binding site did not, however, appear to affect the P50 for oxygen binding.
-
NASA Astrophysics Data System (ADS)
Tanumihardja, J.; Bela, B.
2017-08-01
Fusion inhibitors have potential for future use in HIV control programs in Indonesia, so the capacity to test resistance to such drugs needs to be developed. Resistance-detection with a genotypic assay began with amplification of the target gene, gp41. Based on the sequence of the two most common HIV subtypes in Indonesia, AE and B, a primer pair was designed. Plasma samples containing both subtypes were extracted to obtain HIV RNA. Using PCR, the primer pair was used to produce the amplification product, the identity of which was checked based on length under electrophoresis. Eleven plasma samples were included in this study. One-step PCR using the primer pair was able to amplify gp41 from 54.5% of the samples, and an unspecific amplification product was seen in 1.1% of the samples. Amplification failed in 36.4% of the samples, which may be due to an inappropriate primer sequence. It was also found that the optimal annealing temperature for producing the single expected band was 57.2 °C. With one-step PCR, the designed primer pair amplified the HIV-1 gp41 gene from subtypes AE and B. However, further research should be done to determine the conditions that will increase the sensitivity and specificity of the amplification process.
-
Ferenci, T; Lee, K S
1989-01-01
Maltoporin trimers constitute maltodextrin-selective channels in the outer membrane of Escherichia coli. To study the organization of the maltodextrin-binding site within trimers, dominance studies were undertaken with maltoporin variants of altered binding affinity. It has been established that amino acid substitutions at three dispersed regions of the maltoporin sequence (at residues 8, 82, and 360) resulted specifically in maltodextrin-binding defects and loss of maltodextrin channel selectivity; a substitution at residue 118 increased both binding affinity and maltodextrin transport. Strains heterodiploid for lamB were constructed in which these substitutions were encoded by chromosomal and plasmid-borne genes, and the relative level of maltoporin expression from these genes was estimated. Binding assays with bacteria forming maltoporin heterotrimers were performed in order to test for complementation between binding-negative alleles, negative dominance of negative over wild-type alleles, and possible dominance of negatives over the high-affinity allele. Double mutants with mutations affecting residues 8 and 118, 82 and 118, and 118 and 360 were constructed in vitro, and the dominance properties of the mutations in cis were also tested. There was no complementation between negatives and no negative dominance in heterotrimers. The high-affinity mutation was dominant over negatives in trans but not in cis. The affinity of binding sites in heterotrimer populations was characteristic of the high-affinity allele present and uninfluenced by the negative allele. These results are consistent with the presence of three discrete binding sites in a maltoporin trimer and suggest that the selectivity filter for maltodextrins is not at the interface between the three subunits. PMID:2521623
-
Cardiac muscle activation blunted by a mutation to the regulatory component, troponin T.
Kobayashi, Minae; Debold, Edward P; Turner, Matthew A; Kobayashi, Tomoyoshi
2013-09-06
The striated muscle thin filament comprises actin, tropomyosin, and troponin. The Tn complex consists of three subunits, troponin C (TnC), troponin I (TnI), and troponin T (TnT). TnT may serve as a bridge between the Ca(2+) sensor (TnC) and the actin filament. In the short helix preceding the IT-arm region, H1(T2), there are known dilated cardiomyopathy-linked mutations (among them R205L). Thus we hypothesized that there is an element in this short helix that plays an important role in regulating the muscle contraction, especially in Ca(2+) activation. We mutated Arg-205 and several other amino acid residues within and near the H1(T2) helix. Utilizing an alanine replacement method to compare the effects of the mutations, the biochemical and mechanical impact on the actomyosin interaction was assessed by solution ATPase activity assay, an in vitro motility assay, and Ca(2+) binding measurements. Ca(2+) activation was markedly impaired by a point mutation of the highly conserved basic residue R205A, residing in the short helix H1(T2) of cTnT, whereas the mutations to nearby residues exhibited little effect on function. Interestingly, rigor activation was unchanged between the wild type and R205A TnT. In addition to the reduction in Ca(2+) sensitivity observed in Ca(2+) binding to the thin filament, myosin S1-ADP binding to the thin filament was significantly affected by the same mutation, which was also supported by a series of S1 concentration-dependent ATPase assays. These suggest that the R205A mutation alters function through reduction in the nature of cooperative binding of S1.
-
Structural basis for DNA binding by replication initiator Mcm10
DOE Office of Scientific and Technical Information (OSTI.GOV)
Warren, Eric M.; Vaithiyalingam, Sivaraja; Haworth, Justin
2009-06-30
Mcm10 is an essential eukaryotic DNA replication protein required for assembly and progression of the replication fork. The highly conserved internal domain (Mcm10-ID) has been shown to physically interact with single-stranded (ss) DNA, DNA polymerase alpha, and proliferating cell nuclear antigen (PCNA). The crystal structure of Xenopus laevis Mcm10-ID presented here reveals a DNA binding architecture composed of an oligonucleotide/oligosaccharide-fold followed in tandem by a variant and highly basic zinc finger. NMR chemical shift perturbation and mutational studies of DNA binding activity in vitro reveal how Mcm10 uses this unique surface to engage ssDNA. Corresponding mutations in Saccharomyces cerevisiae resultmore » in increased sensitivity to replication stress, demonstrating the functional importance of DNA binding by this region of Mcm10 to replication. In addition, mapping Mcm10 mutations known to disrupt PCNA, polymerase alpha, and DNA interactions onto the crystal structure provides insight into how Mcm10 might coordinate protein and DNA binding within the replisome.« less
-
Protein-mediated antagonism between HIV reverse transcriptase ligands nevirapine and MgATP.
Zheng, Xunhai; Mueller, Geoffrey A; DeRose, Eugene F; London, Robert E
2013-06-18
Nonnucleoside reverse transcriptase inhibitors (NNRTIs) play a central role in the treatment of AIDS, but their mechanisms of action are incompletely understood. The interaction of the NNRTI nevirapine (NVP) with HIV-1 reverse transcriptase (RT) is characterized by a preference for the open conformation of the fingers/thumb subdomains, and a reported variation of three orders of magnitude between the binding affinity of NVP for RT in the presence or absence of primer/template DNA. To investigate the relationship between conformation and ligand binding, we evaluated the use of methionine NMR probes positioned near the tip of the fingers or thumb subdomains. Such probes would be expected to be sensitive to changes in the local environment depending on the fractions of open and closed RT. Comparisons of the NMR spectra of three conservative mutations, I63M, L74M, and L289M, indicated that M63 showed the greatest shift sensitivity to the addition of NVP. The exchange kinetics of the M63 resonance are fast on the chemical shift timescale, but become slow in the presence of NVP due to the slow binding of RT with the inhibitor. The simplest model consistent with this behavior involves a rapid open/closed equilibrium coupled with a slow interaction of the inhibitor with the open conformation. Studies of RT in the presence of both NVP and MgATP indicate a strong negative cooperativity. Binding of MgATP reduces the fraction of RT bound to NVP, as indicated by the intensity of the NVP-perturbed M230 resonance, and enhances the dissociation rate constant of the NVP, resulting in an increase of the open/closed interconversion rate, so that the M63 resonance moves into the fast/intermediate-exchange regime. Protein-mediated interactions appear to explain most of the affinity variation of NVP for RT. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.
-
[Analysis of gene mutation in a Chinese family with Norrie disease].
Zhang, Tian-xiao; Zhao, Xiu-li; Hua, Rui; Zhang, Jin-song; Zhang, Xue
2012-09-01
To detect the pathogenic mutation in a Chinese family with Norrie disease. Clinical diagnosis was based on familial history, clinical sign and B ultrasonic examination. Peripheral blood samples were obtained from all available members in a Chinese family with Norrie disease. Genomic DNA was extracted from lymphocytes by the standard SDS-proteinase K-phenol/chloroform method. Two coding exons and all intron-exon boundaries of the NDP gene were PCR amplified using three pairs of primers and subjected to automatic DNA sequence. The causative mutation was confirmed by restriction enzyme analysis and genotyping analysis in all members. Sequence analysis of NDP gene revealed a missense mutation c.220C > T (p.Arg74Cys) in the proband and his mother. Further mutation identification by restriction enzyme analysis and genotyping analysis showed that the proband was homozygote of this mutation. His mother and other four unaffected members (III3, IV4, III5 and II2) were carriers of this mutation. The mutant amino acid located in the C-terminal cystine knot-like domain, which was critical motif for the structure and function of NDP. A NDP missense mutation was identified in a Chinese family with Norrie disease.
-
Khordadpoor-Deilamani, Faravareh; Akbari, Mohammad Taghi; Karimipoor, Morteza; Javadi, Gholam Reza
2016-05-01
Albinism is a heterogeneous genetic disorder of melanin synthesis that results in hypopigmented hair, skin and eyes. It is associated with decreased visual acuity, nystagmus, strabismus and photophobia. Six genes are known to be involved in nonsyndromic oculocutaneous albinism (OCA). In this study, we aimed to find the disease causing mutations in albinism patients using homozygosity mapping. Twenty three unrelated patients with nonsyndromic OCA or autosomal recessive ocular albinism were recruited in this study. All of the patients' parents had consanguineous marriage and all were screened for TYR mutations previously. At first, we performed homozygosity mapping using fluorescently labeled primers to amplify a novel panel of 13 STR markers inside the OCA genes and then the screened loci in each family were studied using PCR and cycle sequencing methods. We found five mutations including three mutations in OCA2, one mutation in SLC45A2 and one mutation in C10ORF11 genes, all of which were novel. In cases where the disease causing mutations are identical by descent due to a common ancestor, these STR markers can enable us to screen for the responsible genes.
-
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
-
KMeyeDB: a graphical database of mutations in genes that cause eye diseases.
Kawamura, Takashi; Ohtsubo, Masafumi; Mitsuyama, Susumu; Ohno-Nakamura, Saho; Shimizu, Nobuyoshi; Minoshima, Shinsei
2010-06-01
KMeyeDB (http://mutview.dmb.med.keio.ac.jp/) is a database of human gene mutations that cause eye diseases. We have substantially enriched the amount of data in the database, which now contains information about the mutations of 167 human genes causing eye-related diseases including retinitis pigmentosa, cone-rod dystrophy, night blindness, Oguchi disease, Stargardt disease, macular degeneration, Leber congenital amaurosis, corneal dystrophy, cataract, glaucoma, retinoblastoma, Bardet-Biedl syndrome, and Usher syndrome. KMeyeDB is operated using the database software MutationView, which deals with various characters of mutations, gene structure, protein functional domains, and polymerase chain reaction (PCR) primers, as well as clinical data for each case. Users can access the database using an ordinary Internet browser with smooth user-interface, without user registration. The results are displayed on the graphical windows together with statistical calculations. All mutations and associated data have been collected from published articles. Careful data analysis with KMeyeDB revealed many interesting features regarding the mutations in 167 genes that cause 326 different types of eye diseases. Some genes are involved in multiple types of eye diseases, whereas several eye diseases are caused by different mutations in one gene.
-
Fedele, Laura; Newcombe, Joseph; Topf, Maya; Gibb, Alasdair; Harvey, Robert J; Smart, Trevor G
2018-03-06
Genetic and bioinformatic analyses have identified missense mutations in GRIN2B encoding the NMDA receptor GluN2B subunit in autism, intellectual disability, Lennox Gastaut and West Syndromes. Here, we investigated several such mutations using a near-complete, hybrid 3D model of the human NMDAR and studied their consequences with kinetic modelling and electrophysiology. The mutants revealed reductions in glutamate potency; increased receptor desensitisation; and ablation of voltage-dependent Mg 2+ block. In addition, we provide new views on Mg 2+ and NMDA channel blocker binding sites. We demonstrate that these mutants have significant impact on excitatory transmission in developing neurons, revealing profound changes that could underlie their associated neurological disorders. Of note, the NMDAR channel mutant GluN2B V618G unusually allowed Mg 2+ permeation, whereas nearby N615I reduced Ca 2+ permeability. By identifying the binding site for an NMDAR antagonist that is used in the clinic to rescue gain-of-function phenotypes, we show that drug binding may be modified by some GluN2B disease-causing mutations.
-
Salmas, Ramin Ekhteiari; Mestanoglu, Mert; Unlu, Ayhan; Yurtsever, Mine; Durdagi, Serdar
2016-11-01
Mutated form (G52E) of diphtheria toxin (DT) CRM197 is an inactive and nontoxic enzyme. Here, we provided a molecular insight using comparative molecular dynamics (MD) simulations to clarify the influence of a single point mutation on overall protein and active-site loop. Post-processing MD analysis (i.e. stability, principal component analysis, hydrogen-bond occupancy, etc.) is carried out on both wild and mutated targets to investigate and to better understand the mechanistic differences of structural and dynamical properties on an atomic scale especially at nicotinamide adenine dinucleotide (NAD) binding site when a single mutation (G52E) happens at the DT. In addition, a docking simulation is performed for wild and mutated forms. The docking scoring analysis and docking poses results revealed that mutant form is not able to properly accommodate the NAD molecule.
-
NASA Astrophysics Data System (ADS)
Woods, Christopher J.; Malaisree, Maturos; Long, Ben; McIntosh-Smith, Simon; Mulholland, Adrian J.
2013-12-01
The emergence of a novel H7N9 avian influenza that infects humans is a serious cause for concern. Of the genome sequences of H7N9 neuraminidase available, one contains a substitution of arginine to lysine at position 292, suggesting a potential for reduced drug binding efficacy. We have performed molecular dynamics simulations of oseltamivir, zanamivir and peramivir bound to H7N9, H7N9-R292K, and a structurally related H11N9 neuraminidase. They show that H7N9 neuraminidase is structurally homologous to H11N9, binding the drugs in identical modes. The simulations reveal that the R292K mutation disrupts drug binding in H7N9 in a comparable manner to that observed experimentally for H11N9-R292K. Absolute binding free energy calculations with the WaterSwap method confirm a reduction in binding affinity. This indicates that the efficacy of antiviral drugs against H7N9-R292K will be reduced. Simulations can assist in predicting disruption of binding caused by mutations in neuraminidase, thereby providing a computational `assay.'
-
Enciso-Rodríguez, Felix E.; González, Carolina; Rodríguez, Edwin A.; López, Camilo E.; Landsman, David; Barrero, Luz Stella; Mariño-Ramírez, Leonardo
2013-01-01
The Cape gooseberry ( Physalis peruviana L) is an Andean exotic fruit with high nutritional value and appealing medicinal properties. However, its cultivation faces important phytosanitary problems mainly due to pathogens like Fusarium oxysporum, Cercosporaphysalidis and Alternaria spp. Here we used the Cape gooseberry foliar transcriptome to search for proteins that encode conserved domains related to plant immunity including: NBS (Nucleotide Binding Site), CC (Coiled-Coil), TIR (Toll/Interleukin-1 Receptor). We identified 74 immunity related gene candidates in P . peruviana which have the typical resistance gene (R-gene) architecture, 17 Receptor like kinase (RLKs) candidates related to PAMP-Triggered Immunity (PTI), eight (TIR-NBS-LRR, or TNL) and nine (CC–NBS-LRR, or CNL) candidates related to Effector-Triggered Immunity (ETI) genes among others. These candidate genes were categorized by molecular function (98%), biological process (85%) and cellular component (79%) using gene ontology. Some of the most interesting predicted roles were those associated with binding and transferase activity. We designed 94 primers pairs from the 74 immunity-related genes (IRGs) to amplify the corresponding genomic regions on six genotypes that included resistant and susceptible materials. From these, we selected 17 single band amplicons and sequenced them in 14 F. oxysporum resistant and susceptible genotypes. Sequence polymorphisms were analyzed through preliminary candidate gene association, which allowed the detection of one SNP at the PpIRG-63 marker revealing a nonsynonymous mutation in the predicted LRR domain suggesting functional roles for resistance. PMID:23844210
-
Enciso-Rodríguez, Felix E; González, Carolina; Rodríguez, Edwin A; López, Camilo E; Landsman, David; Barrero, Luz Stella; Mariño-Ramírez, Leonardo
2013-01-01
The Cape gooseberry (Physalisperuviana L) is an Andean exotic fruit with high nutritional value and appealing medicinal properties. However, its cultivation faces important phytosanitary problems mainly due to pathogens like Fusarium oxysporum, Cercosporaphysalidis and Alternaria spp. Here we used the Cape gooseberry foliar transcriptome to search for proteins that encode conserved domains related to plant immunity including: NBS (Nucleotide Binding Site), CC (Coiled-Coil), TIR (Toll/Interleukin-1 Receptor). We identified 74 immunity related gene candidates in P. peruviana which have the typical resistance gene (R-gene) architecture, 17 Receptor like kinase (RLKs) candidates related to PAMP-Triggered Immunity (PTI), eight (TIR-NBS-LRR, or TNL) and nine (CC-NBS-LRR, or CNL) candidates related to Effector-Triggered Immunity (ETI) genes among others. These candidate genes were categorized by molecular function (98%), biological process (85%) and cellular component (79%) using gene ontology. Some of the most interesting predicted roles were those associated with binding and transferase activity. We designed 94 primers pairs from the 74 immunity-related genes (IRGs) to amplify the corresponding genomic regions on six genotypes that included resistant and susceptible materials. From these, we selected 17 single band amplicons and sequenced them in 14 F. oxysporum resistant and susceptible genotypes. Sequence polymorphisms were analyzed through preliminary candidate gene association, which allowed the detection of one SNP at the PpIRG-63 marker revealing a nonsynonymous mutation in the predicted LRR domain suggesting functional roles for resistance.
-
Toral-López, Jaime; González-Huerta, Luz M; Martín-Del Campo, Mónica; Messina-Baas, Olga; Cuevas-Covarrubias, Sergio A
2018-05-01
The proband in this study was a 4-year-old Mexican girl with Blau syndrome. She and her affected family members had skin rash and arthritis but no uveitis. Exome sequencing and DNA direct sequencing from blood samples revealed a novel nucleotide-binding oligomerization domain-containing protein 2 gene mutation in the affected family members. This study is the first report of a Mexican family with Blau syndrome showing good infliximab treatment response. The novel mutation in the nucleotide-binding oligomerization domain-containing protein 2 gene (c.1808A>G) enriches the mutation spectrum in Blau syndrome. This family represents one of the few cases of autosomal Blau syndrome with no uveitis; because of phenotype variability, it is important to recognize Blau syndrome's clinical spectrum and recommend genetic consultation. © 2018 Wiley Periodicals, Inc.
-
2013-01-01
Background Rubinstein-Taybi syndrome (RTS) is a rare autosomal dominant disorder (prevalence 1:125,000) characterised by broad thumbs and halluces, facial dysmorphism, psychomotor development delay, skeletal defects, abnormalities in the posterior fossa and short stature. The known genetic causes are point mutations or deletions of the cAMP-response element binding protein-BP (CREBBP) (50-60% of the cases) and of the homologous gene E1A-binding protein (EP300) (5%). Case presentation We describe, for the first time in literature, a RTS Caucasian girl, 14-year-old, with growth hormone (GH) deficiency, pituitary hypoplasia, Arnold Chiari malformation type 1, double syringomyelic cavity and a novel CREBBP mutation (c.3546insCC). Conclusion We hypothesize that CREBBP mutation we have identified in this patient could be responsible also for RTS atypical features as GH deficiency and pituitary hypoplasia. PMID:23432975
-
Rapid amplification of 5' complementary DNA ends (5' RACE).
2005-08-01
This method is used to extend partial cDNA clones by amplifying the 5' sequences of the corresponding mRNAs 1-3. The technique requires knowledge of only a small region of sequence within the partial cDNA clone. During PCR, the thermostable DNA polymerase is directed to the appropriate target RNA by a single primer derived from the region of known sequence; the second primer required for PCR is complementary to a general feature of the target-in the case of 5' RACE, to a homopolymeric tail added (via terminal transferase) to the 3' termini of cDNAs transcribed from a preparation of mRNA. This synthetic tail provides a primer-binding site upstream of the unknown 5' sequence of the target mRNA. The products of the amplification reaction are cloned into a plasmid vector for sequencing and subsequent manipulation.
-
Singh, O. P.; Dykes, C. L.; Sharma, G.; Das, M. K.
2015-01-01
Leucine-to-phenylalanine substitution at residue L1014 in the voltage-gated sodium channel, target site of action for dichlorodiphenyltrichloroethane (DDT) and pyrethroids, is the most common knockdown resistance (kdr) mutation reported in several insects conferring resistance against DDT and pyrethroids. Here, we report presence of two coexisting alternative transversions, A>T and A>C, on the third codon position of L1014 residue in malaria vector Anopheles subpictus Grassi (species A) from Jamshedpur (India), both leading to the same amino acid substitution of Leu-to-Phe with allelic frequencies of 19 and 67%, respectively. A single primer-introduced restriction analysis–polymerase chain reaction (PIRA-PCR) was devised for the identification of L1014F-kdr mutation in An. subpictus resulting from either type of point mutation. Genotyping of samples with PIRA-PCR revealed high frequency (82%) of L1014F-kdr mutation in the study area. PMID:26336276
-
Zúñiga, Matías A; Alderete, Joel B; Jaña, Gonzalo A; Jiménez, Verónica A
2017-07-01
Peloruside A (PLA) and Laulimalide (LAU) are novel microtubule-stabilizing agents with promising properties against different cancer types. These ligands share a non-taxoid binding site at the outer surface of β-tubulin and promote microtubule stabilization by bridging two adjacent αβ-tubulin dimers from parallel protofilaments. Recent site-directed mutagenesis experiments confirmed the existence of a unique β-tubulin site mutation (Gln293Met) that specifically increased the activity of PLA and caused resistance to LAU, without affecting the stability of microtubules in the absence of the ligands. In this work, fully atomistic molecular dynamics simulations were carried out to examine the PLA and LAU association with native and mutated αβ-tubulin in the search for structural and energetic evidence to explain the role of Gln293Met mutation on determining the activity of these ligands. Our results revealed that Gln293Met mutation induced the loss of relevant LAU-tubulin contacts but exerted negligible changes in the interaction networks responsible for PLA-tubulin association. Binding free energy calculations (MM/GBSA and MM/PBSA), and weak interaction analysis (aNCI) predicted an increased affinity for PLA, and a weakened association for LAU after mutation, thus suggesting that Gln293Met mutation exerts its action by a modulation of drug-tubulin interactions. These results are valuable to increase understanding about PLA and LAU activity and to assist the future design of novel agents targeting the PLA/LAU binding pocket.
-
NASA Astrophysics Data System (ADS)
Zúñiga, Matías A.; Alderete, Joel B.; Jaña, Gonzalo A.; Jiménez, Verónica A.
2017-07-01
Peloruside A (PLA) and Laulimalide (LAU) are novel microtubule-stabilizing agents with promising properties against different cancer types. These ligands share a non-taxoid binding site at the outer surface of β-tubulin and promote microtubule stabilization by bridging two adjacent αβ-tubulin dimers from parallel protofilaments. Recent site-directed mutagenesis experiments confirmed the existence of a unique β-tubulin site mutation (Gln293Met) that specifically increased the activity of PLA and caused resistance to LAU, without affecting the stability of microtubules in the absence of the ligands. In this work, fully atomistic molecular dynamics simulations were carried out to examine the PLA and LAU association with native and mutated αβ-tubulin in the search for structural and energetic evidence to explain the role of Gln293Met mutation on determining the activity of these ligands. Our results revealed that Gln293Met mutation induced the loss of relevant LAU-tubulin contacts but exerted negligible changes in the interaction networks responsible for PLA-tubulin association. Binding free energy calculations (MM/GBSA and MM/PBSA), and weak interaction analysis (aNCI) predicted an increased affinity for PLA, and a weakened association for LAU after mutation, thus suggesting that Gln293Met mutation exerts its action by a modulation of drug-tubulin interactions. These results are valuable to increase understanding about PLA and LAU activity and to assist the future design of novel agents targeting the PLA/LAU binding pocket.
-
STITCHER: A web resource for high-throughput design of primers for overlapping PCR applications.
O'Halloran, Damien M
2015-06-01
Overlapping PCR is routinely used in a wide number of molecular applications. These include stitching PCR fragments together, generating fluorescent transcriptional and translational fusions, inserting mutations, making deletions, and PCR cloning. Overlapping PCR is also used for genotyping by traditional PCR techniques and in detection experiments using techniques such as loop-mediated isothermal amplification (LAMP). STITCHER is a web tool providing a central resource for researchers conducting all types of overlapping PCR experiments with an intuitive interface for automated primer design that's fast, easy to use, and freely available online (http://ohalloranlab.net/STITCHER.html). STITCHER can handle both single sequence and multi-sequence input, and specific features facilitate numerous other PCR applications, including assembly PCR, adapter PCR, and primer walking. Field PCR, and in particular, LAMP, offers promise as an on site tool for pathogen detection in underdeveloped areas, and STITCHER includes off-target detection features for pathogens commonly targeted using LAMP technology.
-
Prats, A C; Sarih, L; Gabus, C; Litvak, S; Keith, G; Darlix, J L
1988-01-01
Retrovirus virions carry a diploid genome associated with a large number of small viral finger protein molecules which are required for encapsidation. Our present results show that finger protein p12 of Rous sarcoma virus (RSV) and p10 of murine leukaemia virus (MuLV) positions replication primer tRNA on the replication initiation site (PBS) at the 5' end of the RNA genome. An RSV mutant with a Val-Pro insertion in the finger motif of p12 is able to partially encapsidate genomic RNA but is not infectious because mutated p12 is incapable of positioning the replication primer, tRNATrp. Since all known replication competent retroviruses, and the plant virus CaMV, code for finger proteins analogous to RSV p12 or MuLV p10, the initial stage of reverse transcription in avian, mammalian and human retroviruses and in CaMV is probably controlled in an analogous way. Images PMID:2458920
-
Prats, A C; Sarih, L; Gabus, C; Litvak, S; Keith, G; Darlix, J L
1988-06-01
Retrovirus virions carry a diploid genome associated with a large number of small viral finger protein molecules which are required for encapsidation. Our present results show that finger protein p12 of Rous sarcoma virus (RSV) and p10 of murine leukaemia virus (MuLV) positions replication primer tRNA on the replication initiation site (PBS) at the 5' end of the RNA genome. An RSV mutant with a Val-Pro insertion in the finger motif of p12 is able to partially encapsidate genomic RNA but is not infectious because mutated p12 is incapable of positioning the replication primer, tRNATrp. Since all known replication competent retroviruses, and the plant virus CaMV, code for finger proteins analogous to RSV p12 or MuLV p10, the initial stage of reverse transcription in avian, mammalian and human retroviruses and in CaMV is probably controlled in an analogous way.
-
Brammer, Leighanne A; Bolduc, Benjamin; Kass, Jessica L; Felice, Kristin M; Noren, Christopher J; Hall, Marilena Fitzsimons
2008-02-01
Screening of the commercially available Ph.D.-7 phage-displayed heptapeptide library for peptides that bind immobilized Zn2+ resulted in the repeated selection of the peptide HAIYPRH, although binding assays indicated that HAIYPRH is not a zinc-binding peptide. HAIYPRH has also been selected in several other laboratories using completely different targets, and its ubiquity suggests that it is a target-unrelated peptide. We demonstrated that phage displaying HAIYPRH are enriched after serial amplification of the library without exposure to target. The amplification of phage displaying HAIYPRH was found to be dramatically faster than that of the library itself. DNA sequencing uncovered a mutation in the Shine-Dalgarno (SD) sequence for gIIp, a protein involved in phage replication, imparting to the SD sequence better complementarity to the 16S ribosomal RNA (rRNA). Introducing this mutation into phage lacking a displayed peptide resulted in accelerated propagation, whereas phage displaying HAIYPRH with a wild-type SD sequence were found to amplify normally. The SD mutation may alter gIIp expression and, consequently, the rate of propagation of phage. In the Ph.D.-7 library, the mutation is coincident with the displayed peptide HAIYPRH, accounting for the target-unrelated selection of this peptide in multiple reported panning experiments.
-
Single-Molecule Counting of Point Mutations by Transient DNA Binding
NASA Astrophysics Data System (ADS)
Su, Xin; Li, Lidan; Wang, Shanshan; Hao, Dandan; Wang, Lei; Yu, Changyuan
2017-03-01
High-confidence detection of point mutations is important for disease diagnosis and clinical practice. Hybridization probes are extensively used, but are hindered by their poor single-nucleotide selectivity. Shortening the length of DNA hybridization probes weakens the stability of the probe-target duplex, leading to transient binding between complementary sequences. The kinetics of probe-target binding events are highly dependent on the number of complementary base pairs. Here, we present a single-molecule assay for point mutation detection based on transient DNA binding and use of total internal reflection fluorescence microscopy. Statistical analysis of single-molecule kinetics enabled us to effectively discriminate between wild type DNA sequences and single-nucleotide variants at the single-molecule level. A higher single-nucleotide discrimination is achieved than in our previous work by optimizing the assay conditions, which is guided by statistical modeling of kinetics with a gamma distribution. The KRAS c.34 A mutation can be clearly differentiated from the wild type sequence (KRAS c.34 G) at a relative abundance as low as 0.01% mutant to WT. To demonstrate the feasibility of this method for analysis of clinically relevant biological samples, we used this technology to detect mutations in single-stranded DNA generated from asymmetric RT-PCR of mRNA from two cancer cell lines.
-
Osawa, Masaki
2018-01-01
It is difficult to target and kill cancer cells. One possible approach is to mutate bacteria to enhance their binding to cancer cells. In the present study, Gram-negative Escherichia coli and Gram-positive Bacillus subtilis were randomly mutated, and then were positively and negatively selected for binding cancer vs normal cells. With repetitive mutation and selection both bacteria successfully evolved to increase affinity to the pancreatic cancer cell line (Mia PaCa-2) but not normal cells (HPDE: immortalized human pancreatic ductal epithelial cells). The mutant E. coli and B. subtilis strains bound to Mia PaCa-2 cells about 10 and 25 times more than to HPDE cells. The selected E. coli strain had mutations in biofilm-related genes and the regulatory region for a type I pilus gene. Consistent with type I pili involvement, mannose could inhibit the binding to cells. The results suggest that weak but specific binding is involved in the initial step of adhesion. To test their ability to kill Mia PaCa-2 cells, hemolysin was expressed in the mutant strain. The hemolysin released from the mutant strain was active and could kill Mia PaCa-2 cells. In the case of B. subtilis, the initial binding to the cells was a weak interaction of the leading pole of the motile bacteria. The frequency of this interaction to Mia PaCa-2 cells dramatically increased in the evolved mutant strain. This mutant strain could also specifically invade beneath Mia PaCa-2 cells and settle there. This type of mutation/selection strategy may be applicable to other combinations of cancer cells and bacterial species.
-
The interplay between effector binding and allostery in an engineered protein switch.
Choi, Jay H; Xiong, Tina; Ostermeier, Marc
2016-09-01
The protein design rules for engineering allosteric regulation are not well understood. A fundamental understanding of the determinants of ligand binding in an allosteric context could facilitate the design and construction of versatile protein switches and biosensors. Here, we conducted extensive in vitro and in vivo characterization of the effects of 285 unique point mutations at 15 residues in the maltose-binding pocket of the maltose-activated β-lactamase MBP317-347. MBP317-347 is an allosteric enzyme formed by the insertion of TEM-1 β-lactamase into the E. coli maltose binding protein (MBP). We find that the maltose-dependent resistance to ampicillin conferred to the cells by the MBP317-347 switch gene (the switch phenotype) is very robust to mutations, with most mutations slightly improving the switch phenotype. We identified 15 mutations that improved switch performance from twofold to 22-fold, primarily by decreasing the catalytic activity in the absence of maltose, perhaps by disrupting interactions that cause a small fraction of MBP in solution to exist in a partially closed state in the absence of maltose. Other notable mutations include K15D and K15H that increased maltose affinity 30-fold and Y155K and Y155R that compromised switching by diminishing the ability of maltose to increase catalytic activity. The data also provided insights into normal MBP physiology, as select mutations at D14, W62, and F156 retained high maltose affinity but abolished the switch's ability to substitute for MBP in the transport of maltose into the cell. The results reveal the complex relationship between ligand binding and allostery in this engineered switch. © 2016 The Protein Society.
-
Machado, Danielle S.; Sabet, Amin; Santiago, Leticia A.; Sidhaye, Aniket R.; Chiamolera, Maria I.; Ortiga-Carvalho, Tania M.; Wondisford, Fredric E.
2009-01-01
Resistance to thyroid hormone (RTH) is most often due to point mutations in the β-isoform of the thyroid hormone (TH) receptor (TR-β). The majority of mutations involve the ligand-binding domain, where they block TH binding and receptor function on both stimulatory and inhibitory TH response elements. In contrast, a few mutations in the ligand-binding domain are reported to maintain TH binding and yet cause RTH in certain tissues. We introduced one such naturally occurring human RTH mutation (R429Q) into the germline of mice at the TR-β locus. R429Q knock-in (KI) mice demonstrated elevated serum TH and inappropriately normal thyroid-stimulating hormone (TSH) levels, consistent with hypothalamic–pituitary RTH. In contrast, 3 hepatic genes positively regulated by TH (Dio1, Gpd1, and Thrsp) were increased in R429Q KI animals. Mice were then rendered hypothyroid, followed by graded T3 replacement. Hypothyroid R429Q KI mice displayed elevated TSH subunit mRNA levels, and T3 treatment failed to normally suppress these levels. T3 treatment, however, stimulated pituitary Gh levels to a greater degree in R429Q KI than in control mice. Gsta, a hepatic gene negatively regulated by TH, was not suppressed in R429Q KI mice after T3 treatment, but hepatic Dio1 and Thrsp mRNA levels increased in response to TH. Cardiac myosin heavy chain isoform gene expression also showed a specific defect in TH inhibition. In summary, the R429Q mutation is associated with selective impairment of TH-mediated gene repression, suggesting that the affected domain, necessary for TR homodimerization and corepressor binding, has a critical role in negative gene regulation by TH. PMID:19439650
-
Signorelli, Sara; Santini, Simona; Yamada, Tohru; Bizzarri, Anna Rita; Beattie, Craig W; Cannistraro, Salvatore
2017-04-01
Mutations within the DNA binding domain (DBD) of the tumor suppressor p53 are found in >50% of human cancers and may significantly modify p53 secondary structure impairing its function. p28, an amphipathic cell-penetrating peptide, binds to the DBD through hydrophobic interaction and induces a posttranslational increase in wildtype and mutant p53 restoring functionality. We use mutation analyses to explore which elements of secondary structure may be critical to p28 binding. Molecular modeling, Raman spectroscopy, Atomic Force Spectroscopy (AFS) and Surface Plasmon Resonance (SPR) were used to identify which secondary structure of site-directed and naturally occurring mutant DBDs are potentially altered by discrete changes in hydrophobicity and the molecular interaction with p28. We show that specific point mutations that alter hydrophobicity within non-mutable and mutable regions of the p53 DBD alter specific secondary structures. The affinity of p28 was positively correlated with the β-sheet content of a mutant DBD, and reduced by an increase in unstructured or random coil that resulted from a loss in hydrophobicity and redistribution of surface charge. These results help refine our knowledge of how mutations within p53-DBD alter secondary structure and provide insight on how potential structural alterations in p28 or similar molecules improve their ability to restore p53 function. Raman spectroscopy, AFS, SPR and computational modeling are useful approaches to characterize how mutations within the p53DBD potentially affect secondary structure and identify those structural elements prone to influence the binding affinity of agents designed to increase the functionality of p53. Copyright © 2017 Elsevier B.V. All rights reserved.
-
Koenig, Patrick; Sanowar, Sarah; Lee, Chingwei V; Fuh, Germaine
Monoclonal antibodies developed for therapeutic or diagnostic purposes need to demonstrate highly defined binding specificity profiles. Engineering of an antibody to enhance or reduce binding to related antigens is often needed to achieve the desired biologic activity without safety concern. Here, we describe a deep sequencing-aided engineering strategy to fine-tune the specificity of an angiopoietin-2 (Ang2)/vascular endothelial growth factor (VEGF) dual action Fab, 5A12.1 for the treatment of age-related macular degeneration. This antibody utilizes overlapping complementarity-determining region (CDR) sites for dual Ang2/VEGF interaction with K D in the sub-nanomolar range. However, it also exhibits significant (K D of 4 nM) binding to angiopoietin-1, which has high sequence identity with Ang2. We generated a large phage-displayed library of 5A12.1 Fab variants with all possible single mutations in the 6 CDRs. By tracking the change of prevalence of each mutation during various selection conditions, we identified 35 mutations predicted to decrease the affinity for Ang1 while maintaining the affinity for Ang2 and VEGF. We confirmed the specificity profiles for 25 of these single mutations as Fab protein. Structural analysis showed that some of the Fab mutations cluster near a potential Ang1/2 epitope residue that differs in the 2 proteins, while others are up to 15 Å away from the antigen-binding site and likely influence the binding interaction remotely. The approach presented here provides a robust and efficient method for specificity engineering that does not require prior knowledge of the antigen antibody interaction and can be broadly applied to antibody specificity engineering projects.
-
Lu, Shaoyong; Banerjee, Avik; Jang, Hyunbum; Zhang, Jian; Gaponenko, Vadim; Nussinov, Ruth
2015-01-01
K-Ras4B, a frequently mutated oncogene in cancer, plays an essential role in cell growth, differentiation, and survival. Its C-terminal membrane-associated hypervariable region (HVR) is required for full biological activity. In the active GTP-bound state, the HVR interacts with acidic plasma membrane (PM) headgroups, whereas the farnesyl anchors in the membrane; in the inactive GDP-bound state, the HVR may interact with both the PM and the catalytic domain at the effector binding region, obstructing signaling and nucleotide exchange. Here, using molecular dynamics simulations and NMR, we aim to figure out the effects of nucleotides (GTP and GDP) and frequent (G12C, G12D, G12V, G13D, and Q61H) and infrequent (E37K and R164Q) oncogenic mutations on full-length K-Ras4B. The mutations are away from or directly at the HVR switch I/effector binding site. Our results suggest that full-length wild-type GDP-bound K-Ras4B (K-Ras4BWT-GDP) is in an intrinsically autoinhibited state via tight HVR-catalytic domain interactions. The looser association in K-Ras4BWT-GTP may release the HVR. Some of the oncogenic mutations weaken the HVR-catalytic domain association in the K-Ras4B-GDP/-GTP bound states, which may facilitate the HVR disassociation in a nucleotide-independent manner, thereby up-regulating oncogenic Ras signaling. Thus, our results suggest that mutations can exert their effects in more than one way, abolishing GTP hydrolysis and facilitating effector binding. PMID:26453300
-
Vasavi, C S; Tamizhselvi, Ramasamy; Munusami, Punnagai
2017-08-01
HIV-1 protease plays a crucial role in viral replication and maturation, which makes it one of the most attractive targets for anti-retroviral therapy. The majority of HIV infections in developing countries are due to non-B subtype. Subtype AE is spreading rapidly and infecting huge population worldwide. The mutations in the active site of subtype AE directly impair the interactions with the inhibitor. The non-active site mutations influence the binding of the inhibitor indirectly and their resistance mechanism is not well understood. It is important to design new effective inhibitors that combat drug resistance in subtype AE protease. In this work, we examined the effect of non active site mutations L10F, L10F/N88S and L90M with nelfinavir using molecular dynamics simulation and binding free energy calculations. The simulations suggested that the L10F and L10F/N88S mutants decrease the binding affinity of nelfinavir, whereas the L90M mutant increases the binding affinity. The formation of hydrogen bonds between nelfinavir and Asp30 is crucial for effective binding. The benzamide moiety of nelfinavir shows large positional deviation in L10F and L10F/N88S complexes and the L10F/N88S mutation changes the hydrogen bond between the side chain atoms of 30th residue and the 88th residue. Consequently the hydrogen bond interaction between Asp30 and nelfinavir are destroyed leading to drug resistance. Our present study shed light on the resistance mechanism of the strongly linked mutation L10F/N88S observed experimentally in AE subtype. Copyright © 2017 Elsevier Inc. All rights reserved.
-
The Binding Site of Human Adenosine Deaminase for Cd26/Dipeptidyl Peptidase IV
Richard, Eva; Arredondo-Vega, Francisco X.; Santisteban, Ines; Kelly, Susan J.; Patel, Dhavalkumar D.; Hershfield, Michael S.
2000-01-01
Human, but not murine, adenosine deaminase (ADA) forms a complex with the cell membrane protein CD26/dipeptidyl peptidase IV. CD26-bound ADA has been postulated to regulate extracellular adenosine levels and to modulate the costimulatory function of CD26 on T lymphocytes. Absence of ADA–CD26 binding has been implicated in causing severe combined immunodeficiency due to ADA deficiency. Using human–mouse ADA hybrids and ADA point mutants, we have localized the amino acids critical for CD26 binding to the helical segment 126–143. Arg142 in human ADA and Gln142 in mouse ADA largely determine the capacity to bind CD26. Recombinant human ADA bearing the R142Q mutation had normal catalytic activity per molecule, but markedly impaired binding to a CD26+ ADA-deficient human T cell line. Reduced CD26 binding was also found with ADA from red cells and T cells of a healthy individual whose only expressed ADA has the R142Q mutation. Conversely, ADA with the E217K active site mutation, the only ADA expressed by a severely immunodeficient patient, showed normal CD26 binding. These findings argue that ADA binding to CD26 is not essential for immune function in humans. PMID:11067872
-
Shahin, Hashem; Walsh, Tom; Sobe, Tama; Abu Sa’ed, Judeh; Abu Rayan, Amal; Lynch, Eric D.; Lee, Ming K.; Avraham, Karen B.; King, Mary-Claire; Kanaan, Moein
2006-01-01
In a large consanguineous Palestinian kindred, we previously mapped DFNB28—a locus associated with recessively inherited, prelingual, profound sensorineural hearing impairment—to chromosome 22q13.1. We report here that mutations in a novel 218-kDa isoform of TRIOBP (TRIO and filamentous actin [F-actin] binding protein) are associated with DFNB28 hearing loss in a total of nine Palestinian families. Two nonsense mutations (R347X and Q581X) truncate the protein, and a potentially deleterious missense mutation (G1019R) occurs in a conserved motif in a putative SH3-binding domain. In seven families, 27 deaf individuals are homozygous for one of the nonsense mutations; in two other families, 3 deaf individuals are compound heterozygous for the two nonsense mutations or for Q581X and G1019R. The novel long isoform of TRIOBP has a restricted expression profile, including cochlea, retina, and fetal brain, whereas the original short isoform is widely expressed. Antibodies to TRIOBP reveal expression in sensory cells of the inner ear and colocalization with F-actin along the length of the stereocilia. PMID:16385458
-
DOE Office of Scientific and Technical Information (OSTI.GOV)
Proudfoot, Andrew; Axelrod, Herbert L.; Geralt, Michael
The Dlx5 homeodomain is a transcription factor related to the Drosophila Distal-less gene that is associated with breast and lung cancer, lymphoma, Rett syndrome and osteoporosis in humans. Mutations in the DLX5 gene have been linked to deficiencies in craniofacial and limb development in higher eukaryotes, including Split Hand and Foot Malformation-1 (SHFM-1) in humans. Our characterization of a Dlx5 homeodomain–(CGACTAATTAGTCG) 2 complex by NMR spectroscopy paved the way for determination of its crystal structure at 1.85 Å resolution that enabled rationalization of the effects of disease-related mutations on the protein function. A remarkably subtle mutation, Q186H, is linked tomore » SHFM-1; this change likely affects affinity of DNA binding by disrupting water-mediated interactions with the DNA major groove. A more subtle effect is implicated for the Q178P mutation, which is not in direct contact with the DNA. Our data indicate that these mutations diminish the ability of the Dlx5 homeodomain to recognize and bind target DNAs, and likely destabilize the formation of functional complexes.« less
-
Proudfoot, Andrew; Axelrod, Herbert L.; Geralt, Michael; ...
2016-01-29
The Dlx5 homeodomain is a transcription factor related to the Drosophila Distal-less gene that is associated with breast and lung cancer, lymphoma, Rett syndrome and osteoporosis in humans. Mutations in the DLX5 gene have been linked to deficiencies in craniofacial and limb development in higher eukaryotes, including Split Hand and Foot Malformation-1 (SHFM-1) in humans. Our characterization of a Dlx5 homeodomain–(CGACTAATTAGTCG) 2 complex by NMR spectroscopy paved the way for determination of its crystal structure at 1.85 Å resolution that enabled rationalization of the effects of disease-related mutations on the protein function. A remarkably subtle mutation, Q186H, is linked tomore » SHFM-1; this change likely affects affinity of DNA binding by disrupting water-mediated interactions with the DNA major groove. A more subtle effect is implicated for the Q178P mutation, which is not in direct contact with the DNA. Our data indicate that these mutations diminish the ability of the Dlx5 homeodomain to recognize and bind target DNAs, and likely destabilize the formation of functional complexes.« less
-
Doolan, Kyle M; Colby, David W
2015-01-30
Prion diseases are caused by a structural rearrangement of the cellular prion protein, PrP(C), into a disease-associated conformation, PrP(Sc), which may be distinguished from one another using conformation-specific antibodies. We used mutational scanning by cell-surface display to screen 1341 PrP single point mutants for attenuated interaction with four anti-PrP antibodies, including several with conformational specificity. Single-molecule real-time gene sequencing was used to quantify enrichment of mutants, returning 26,000 high-quality full-length reads for each screened population on average. Relative enrichment of mutants correlated to the magnitude of the change in binding affinity. Mutations that diminished binding of the antibody ICSM18 represented the core of contact residues in the published crystal structure of its complex. A similarly located binding site was identified for D18, comprising discontinuous residues in helix 1 of PrP, brought into close proximity to one another only when the alpha helix is intact. The specificity of these antibodies for the normal form of PrP likely arises from loss of this conformational feature after conversion to the disease-associated form. Intriguingly, 6H4 binding was found to depend on interaction with the same residues, among others, suggesting that its ability to recognize both forms of PrP depends on a structural rearrangement of the antigen. The application of mutational scanning and deep sequencing provides residue-level resolution of positions in the protein-protein interaction interface that are critical for binding, as well as a quantitative measure of the impact of mutations on binding affinity. Copyright © 2014 Elsevier Ltd. All rights reserved.
-
Ngadjeua, Flora; Chiaravalli, Jeanne; Traincard, François; Raynal, Bertrand; Fontan, Elisabeth; Agou, Fabrice
2013-01-01
Hypomorphic mutations in the X-linked human NEMO gene result in various forms of anhidrotic ectodermal dysplasia with immunodeficiency. NEMO function is mediated by two distal ubiquitin binding domains located in the regulatory C-terminal domain of the protein: the coiled-coil 2-leucine zipper (CC2-LZ) domain and the zinc finger (ZF) domain. Here, we investigated the effect of the D406V mutation found in the NEMO ZF of an ectodermal dysplasia with immunodeficiency patients. This point mutation does not impair the folding of NEMO ZF or mono-ubiquitin binding but is sufficient to alter NEMO function, as NEMO-deficient fibroblasts and Jurkat T lymphocytes reconstituted with full-length D406V NEMO lead to partial and strong defects in NF-κB activation, respectively. To further characterize the ubiquitin binding properties of NEMO ZF, we employed di-ubiquitin (di-Ub) chains composed of several different linkages (Lys-48, Lys-63, and linear (Met-1-linked)). We showed that the pathogenic mutation preferentially impairs the interaction with Lys-63 and Met-1-linked di-Ub, which correlates with its ubiquitin binding defect in vivo. Furthermore, sedimentation velocity and gel filtration showed that NEMO ZF, like other NEMO related-ZFs, binds mono-Ub and di-Ub with distinct stoichiometries, indicating the presence of a new Ub site within the NEMO ZF. Extensive mutagenesis was then performed on NEMO ZF and characterization of mutants allowed the proposal of a structural model of NEMO ZF in interaction with a Lys-63 di-Ub chain. PMID:24100029
-
Soparkar, Ketaki; Kinana, Alfred D.; Weeks, Jon W.; Morrison, Keith D.; Nikaido, Hiroshi
2015-01-01
ABSTRACT The AcrB protein of Escherichia coli, together with TolC and AcrA, forms a contiguous envelope conduit for the capture and extrusion of diverse antibiotics and cellular metabolites. In this study, we sought to expand our knowledge of AcrB by conducting genetic and functional analyses. We began with an AcrB mutant bearing an F610A substitution in the drug binding pocket and obtained second-site substitutions that overcame the antibiotic hypersusceptibility phenotype conferred by the F610A mutation. Five of the seven unique single amino acid substitutions—Y49S, V127A, V127G, D153E, and G288C—mapped in the periplasmic porter domain of AcrB, with the D153E and G288C mutations mapping near and at the distal drug binding pocket, respectively. The other two substitutions—F453C and L486W—were mapped to transmembrane (TM) helices 5 and 6, respectively. The nitrocefin efflux kinetics data suggested that all periplasmic suppressors significantly restored nitrocefin binding affinity impaired by the F610A mutation. Surprisingly, despite increasing MICs of tested antibiotics and the efflux of N-phenyl-1-naphthylamine, the TM suppressors did not improve the nitrocefin efflux kinetics. These data suggest that the periplasmic substitutions act by influencing drug binding affinities for the distal binding pocket, whereas the TM substitutions may indirectly affect the conformational dynamics of the drug binding domain. IMPORTANCE The AcrB protein and its homologues confer multidrug resistance in many important human bacterial pathogens. A greater understanding of how these efflux pump proteins function will lead to the development of effective inhibitors against them. The research presented in this paper investigates drug binding pocket mutants of AcrB through the isolation and characterization of intragenic suppressor mutations that overcome the drug susceptibility phenotype of mutations affecting the drug binding pocket. The data reveal a remarkable structure-function plasticity of the AcrB protein pertaining to its drug efflux activity. PMID:26240069
-
Ahmad, N N; McDonald-McGinn, D M; Zackai, E H; Knowlton, R G; LaRossa, D; DiMascio, J; Prockop, D J
1993-01-01
Genetic linkage analyses suggest that mutations in type II collagen may be responsible for Stickler syndrome, or arthro-ophthalmopathy (AO), in many families. In the present study oligonucleotide primers were developed to amplify and directly sequence eight of the first nine exons of the gene for type II procollagen (COL2A1). Analysis of the eight exons in 10 unrelated probands with AO revealed that one had a single-base mutation in one allele that changed the codon of -CGA- for arginine at amino acid position alpha 1-9 in exon 7 to a premature termination signal for translation. The second mutation found to cause AO was, therefore, similar to the first in that both created premature termination signals in the COL2A1 gene. Since mutations producing premature termination signals have not previously been detected in genes for fibrillar collagens, the results raise the possibility that such mutations in the COL2A1 gene are a common cause of AO. Images Figure 2 Figure 3 PMID:8434604
-
Ikenaga, Makoto; Tabuchi, Masakazu; Kawauchi, Tomohiro; Sakai, Masao
2016-09-29
The simultaneous extraction of host plant DNA severely limits investigations of the community structures of plant-associated fungi due to the similar homologies of sequences in primer-annealing positions between fungi and host plants. Although fungal-specific primers have been designed, plant DNA continues to be excessively amplified by PCR, resulting in the underestimation of community structures. In order to overcome this limitation, locked nucleic acid (LNA) primers and PCR clamping by LNA oligonucleotides have been applied to enhance the amplification of fungal internal transcribed spacer (ITS) regions. LNA primers were designed by converting DNA into LNA, which is specific to fungi, at the forward primer side. LNA oligonucleotides, the sequences of which are complementary to the host plants, were designed by overlapping a few bases with the annealing position of the reverse primer. Plant-specific DNA was then converted into LNA at the shifted position from the 3' end of the primer-binding position. PCR using the LNA technique enhanced the amplification of fungal ITS regions, whereas those of the host plants were more likely to be amplified without the LNA technique. A denaturing gradient gel electrophoresis (DGGE) analysis displayed patterns that reached an acceptable level for investigating the community structures of plant-associated fungi using the LNA technique. The sequences of the bands detected using the LNA technique were mostly affiliated with known isolates. However, some sequences showed low similarities, indicating the potential to identify novel fungi. Thus, the application of the LNA technique is considered effective for widening the scope of community analyses of plant-associated fungi.
-
Recurrent and functional regulatory mutations in breast cancer.
Rheinbay, Esther; Parasuraman, Prasanna; Grimsby, Jonna; Tiao, Grace; Engreitz, Jesse M; Kim, Jaegil; Lawrence, Michael S; Taylor-Weiner, Amaro; Rodriguez-Cuevas, Sergio; Rosenberg, Mara; Hess, Julian; Stewart, Chip; Maruvka, Yosef E; Stojanov, Petar; Cortes, Maria L; Seepo, Sara; Cibulskis, Carrie; Tracy, Adam; Pugh, Trevor J; Lee, Jesse; Zheng, Zongli; Ellisen, Leif W; Iafrate, A John; Boehm, Jesse S; Gabriel, Stacey B; Meyerson, Matthew; Golub, Todd R; Baselga, Jose; Hidalgo-Miranda, Alfredo; Shioda, Toshi; Bernards, Andre; Lander, Eric S; Getz, Gad
2017-07-06
Genomic analysis of tumours has led to the identification of hundreds of cancer genes on the basis of the presence of mutations in protein-coding regions. By contrast, much less is known about cancer-causing mutations in non-coding regions. Here we perform deep sequencing in 360 primary breast cancers and develop computational methods to identify significantly mutated promoters. Clear signals are found in the promoters of three genes. FOXA1, a known driver of hormone-receptor positive breast cancer, harbours a mutational hotspot in its promoter leading to overexpression through increased E2F binding. RMRP and NEAT1, two non-coding RNA genes, carry mutations that affect protein binding to their promoters and alter expression levels. Our study shows that promoter regions harbour recurrent mutations in cancer with functional consequences and that the mutations occur at similar frequencies as in coding regions. Power analyses indicate that more such regions remain to be discovered through deep sequencing of adequately sized cohorts of patients.
-
2003-03-14
streptococcal superantigen binding to MHCII on the surface of cells (7–9), suggesting an essential role in both MHCII molecular recognition and TCR-mediated...extent, mutations of side chains found in a second conserved MHCII alpha-chain-binding site consisting of a hydrophobic surface loop decreased T-cell...fraction of dimer is present at T-cell stimulatory concentrations of Spe-C following mutation of the unpaired side chain of cys- teine at residue 27 to
-
Camargo, Hendricka; Nusspaumer, Gretel; Abia, David; Briceño, Verónica; Remacha, Miguel; Ballesta, Juan P G
2011-05-01
The eukaryotic ribosomal proteins P1 and P2 bind to protein P0 through their N-terminal domain to form the essential ribosomal stalk. A mutational analysis points to amino acids at positions 2 and 3 as determinants for the drastic difference of Saccharomyces cerevisiae P1 and P2 half-life, and suggest different degradation mechanisms for each protein type. Moreover, the capacity to form P1/P2 heterodimers is drastically affected by mutations in the P2β four initial amino acids, while these mutations have no effect on P1β. Binding of P2β and, to a lesser extent, P1β to the ribosome is also seriously affected showing the high relevance of the amino acids in the first turn of the NTD α-helix 1 for the stalk assembly. The negative effect of some mutations on ribosome binding can be reversed by the presence of the second P1/P2 couple in the ribosome, indicating a stabilizing structural influence between the two heterodimers. Unexpectedly, some mutations totally abolish heterodimer formation but allow significant ribosome binding and, therefore, a previous P1 and P2 association seems not to be an absolute requirement for stalk assembly. Homology modeling of the protein complexes suggests that the mutated residues can affect the overall protein conformation. © The Author(s) 2011. Published by Oxford University Press.
-
Glycosylation of Cblns attenuates their receptor binding.
Rong, Yongqi; Bansal, Parmil K; Wei, Peng; Guo, Hong; Correia, Kristen; Parris, Jennifer; Morgan, James I
2018-05-18
Cbln1 is the prototype of a family (Cbln1-Cbln4) of secreted glycoproteins and is essential for normal synapse structure and function in cerebellum by bridging presynaptic Nrxn to postsynaptic Grid2. Here we report the effects of glycosylation on the in vitro receptor binding properties of Cblns. Cbln1, 2 and 4 harbor two N-linked glycosylation sites, one at the N-terminus is in a region implicated in Nrxn binding and the second is in the C1q domain, a region involved in Grid2 binding. Mutation (asparagine to glutamine) of the N-terminal site, increased neurexin binding whereas mutation of the C1q site markedly increased Grid2 binding. These mutations did not influence subunit composition of Cbln trimeric complexes (mediated through the C1q domain) nor their assembly into hexamers (mediated by the N-terminal region). Therefore, glycosylation likely masks the receptor binding interfaces of Cblns. As Cbln4 has undetectable Grid2 binding in vitro we assessed whether transgenic expression of wild type Cbln4 or its glycosylation mutants rescued the Cbln1-null phenotype in vivo. Cbln4 partially rescued and both glycosylation mutants completely rescued ataxia in cbln1-null mice. Thus Cbln4 has intrinsic Grid2 binding that is attenuated by glycosylation, and glycosylation mutants exhibit gain of function in vivo. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.
-
Myelin protein zero gene sequencing diagnoses Charcot-Marie-Tooth Type 1B disease
DOE Office of Scientific and Technical Information (OSTI.GOV)
Su, Y.; Zhang, H.; Madrid, R.
1994-09-01
Charcot-Marie-Tooth disease (CMT), the most common genetic neuropathy, affects about 1 in 2600 people in Norway and is found worldwide. CMT Type 1 (CMT1) has slow nerve conduction with demyelinated Schwann cells. Autosomal dominant CMT Type 1B (CMT1B) results from mutations in the myelin protein zero gene which directs the synthesis of more than half of all Schwann cell protein. This gene was mapped to the chromosome 1q22-1q23.1 borderline by fluorescence in situ hybridization. The first 7 of 7 reported CMT1B mutations are unique. Thus the most effective means to identify CMT1B mutations in at-risk family members and fetuses ismore » to sequence the entire coding sequence in dominant or sporadic CMT patients without the CMT1A duplication. Of the 19 primers used in 16 pars to uniquely amplify the entire MPZ coding sequence, 6 primer pairs were used to amplify and sequence the 6 exons. The DyeDeoxy Terminator cycle sequencing method used with four different color fluorescent lables was superior to manual sequencing because it sequences more bases unambiguously from extracted genomic DNA samples within 24 hours. This protocol was used to test 28 CMT and Dejerine-Sottas patients without CMT1A gene duplication. Sequencing MPZ gene-specific amplified fragments identified 9 polymorphic sites within the 6 exons that encode the 248 amino acid MPZ protein. The large number of major CMT1B mutations identified by single strand sequencing are being verified by reverse strand sequencing and when possible, by restriction enzyme analysis. This protocol can be used to distringuish CMT1B patients from othre CMT phenotypes and to determine the CMT1B status of relatives both presymptomatically and prenatally.« less
-
Concerted formation of macromolecular Suppressor–mutator transposition complexes
Raina, Ramesh; Schläppi, Michael; Karunanandaa, Balasulojini; Elhofy, Adam; Fedoroff, Nina
1998-01-01
Transposition of the maize Suppressor–mutator (Spm) transposon requires two element-encoded proteins, TnpA and TnpD. Although there are multiple TnpA binding sites near each element end, binding of TnpA to DNA is not cooperative, and the binding affinity is not markedly affected by the number of binding sites per DNA fragment. However, intermolecular complexes form cooperatively between DNA fragments with three or more TnpA binding sites. TnpD, itself not a sequence-specific DNA-binding protein, binds to TnpA and stabilizes the TnpA–DNA complex. The high redundancy of TnpA binding sites at both element ends and the protein–protein interactions between DNA-bound TnpA complexes and between these and TnpD imply a concerted transition of the element from a linear to a protein crosslinked transposition complex within a very narrow protein concentration range. PMID:9671711
-
Gomez-Hurtado, Nieves; Boczek, Nicole J.; Kryshtal, Dmytro O.; Johnson, Christopher N.; Sun, Jennifer; Nitu, Florentin R.; Cornea, Razvan L.; Chazin, Walter J.; Calvert, Melissa L.; Tester, David J.; Ackerman, Michael J.; Knollmann, Bjorn C.
2016-01-01
Background Calmodulin (CaM) mutations are associated with severe forms of long QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT). We recently reported that CaM mutations were found in 13% of genotype-negative LQTS patients, but the prevalence of CaM mutations in genotype-negative CPVT patients is unknown. Here, we identify and characterize CaM mutations in 12 patients with genotype-negative but clinically-diagnosed CPVT. Methods and Results Mutational analysis of CALM1, CALM2 and CALM3 coding regions, in vitro measurement of CaM-Ca2+ (Ca) binding affinity, RyR2-CaM binding, Ca handling, L-type Ca current (LTCC) and action potential duration (APD). We identified a novel CaM mutation – A103V – in CALM3 in 1 of 12 patients (8%), a female who experienced episodes of exertion-induced syncope since age 10, had normal QT interval, and displayed ventricular ectopy during stress testing consistent with CPVT. A103V modestly lowered CaM Ca-binding affinity (3-fold reduction vs WT-CaM), but did not alter CaM binding to RyR2. In permeabilized cardiomyocytes, A103V-CaM (100 nM) promoted spontaneous Ca wave and spark activity, a cellular phenotype of RyR2 activation. Even a 1:3 mixture of A103V-CaM:WT-CaM activated Ca waves, demonstrating functional dominance. Compared to LQTS D96V-CaM, A103V-CaM had significantly less effects on LTCC inactivation and APD, and caused delayed after depolarizations (DADs) and triggered beats in intact cardiomyocytes. Conclusions We discovered a novel CPVT mutation in the CALM3 gene that shares functional characteristics with established CPVT-associated mutations in CALM1. A small proportion of A103V-CaM is sufficient to evoke arrhythmogenic Ca disturbances via RyR2 dysregulation, which explains the autosomal dominant inheritance. PMID:27516456
-
Hamed, Mazen Y; Arya, Gaurav
2016-05-01
Energy calculations based on MM-GBSA were employed to study various zinc finger protein (ZF) motifs binding to DNA. Mutants of both the DNA bound to their specific amino acids were studied. Calculated energies gave evidence for a relationship between binding energy and affinity of ZF motifs to their sites on DNA. ΔG values were -15.82(12), -3.66(12), and -12.14(11.6) kcal/mol for finger one, finger two, and finger three, respectively. The mutations in the DNA bases reduced the value of the negative energies of binding (maximum value for ΔΔG = 42Kcal/mol for F1 when GCG mutated to GGG, and ΔΔG = 22 kcal/mol for F2, the loss in total energy of binding originated in the loss in electrostatic energies upon mutation (r = .98). The mutations in key amino acids in the ZF motif in positions-1, 2, 3, and 6 showed reduced binding energies to DNA with correlation coefficients between total free energy and electrostatic was .99 and with Van der Waal was .93. Results agree with experimentally found selectivity which showed that Arginine in position-1 is specific to G, while Aspartic acid (D) in position 2 plays a complicated role in binding. There is a correlation between the MD calculated free energies of binding and those obtained experimentally for prepared ZF motifs bound to triplet bases in other reports (), our results may help in the design of ZF motifs based on the established recognition codes based on energies and contributing energies to the total energy.
-
Dinitroanilines Bind α-Tubulin to Disrupt Microtubules
Morrissette, Naomi S.; Mitra, Arpita; Sept, David; Sibley, L. David
2004-01-01
Protozoan parasites are remarkably sensitive to dinitroanilines such as oryzalin, which disrupt plant but not animal microtubules. To explore the basis of dinitroaniline action, we isolated 49 independent resistant Toxoplasma gondii lines after chemical mutagenesis. All 23 of the lines that we examined harbored single point mutations in α-tubulin. These point mutations were sufficient to confer resistance when transfected into wild-type parasites. Several mutations were in the M or N loops, which coordinate protofilament interactions in the microtubule, but most of the mutations were in the core of α-tubulin. Docking studies predict that oryzalin binds with an average affinity of 23 nM to a site located beneath the N loop of Toxoplasma α-tubulin. This binding site included residues that were mutated in several resistant lines. Moreover, parallel analysis of Bos taurus α-tubulin indicated that oryzalin did not interact with this site and had a significantly decreased, nonspecific affinity for vertebrate α-tubulin. We propose that the dinitroanilines act through a novel mechanism, by disrupting M-N loop contacts. These compounds also represent the first class of drugs that act on α-tubulin function. PMID:14742718
-
BeAtMuSiC: Prediction of changes in protein-protein binding affinity on mutations.
Dehouck, Yves; Kwasigroch, Jean Marc; Rooman, Marianne; Gilis, Dimitri
2013-07-01
The ability of proteins to establish highly selective interactions with a variety of (macro)molecular partners is a crucial prerequisite to the realization of their biological functions. The availability of computational tools to evaluate the impact of mutations on protein-protein binding can therefore be valuable in a wide range of industrial and biomedical applications, and help rationalize the consequences of non-synonymous single-nucleotide polymorphisms. BeAtMuSiC (http://babylone.ulb.ac.be/beatmusic) is a coarse-grained predictor of the changes in binding free energy induced by point mutations. It relies on a set of statistical potentials derived from known protein structures, and combines the effect of the mutation on the strength of the interactions at the interface, and on the overall stability of the complex. The BeAtMuSiC server requires as input the structure of the protein-protein complex, and gives the possibility to assess rapidly all possible mutations in a protein chain or at the interface, with predictive performances that are in line with the best current methodologies.
-
DOE Office of Scientific and Technical Information (OSTI.GOV)
Fan, L.; Fuss, J.O.; Cheng, Q.J.
2009-05-18
Mutations in XPD helicase, required for nucleotide excision repair (NER) as part of the transcription/repair complex TFIIH, cause three distinct phenotypes: cancer-prone xeroderma pigmentosum (XP), or aging disorders Cockayne syndrome (CS), and trichothiodystrophy (TTD). To clarify molecular differences underlying these diseases, we determined crystal structures of the XPD catalytic core from Sulfolobus acidocaldarius and measured mutant enzyme activities. Substrate-binding grooves separate adjacent Rad51/RecA-like helicase domains (HD1, HD2) and an arch formed by 4FeS and Arch domains. XP mutations map along the HD1 ATP-binding edge and HD2 DNA-binding channel and impair helicase activity essential for NER. XP/CS mutations both impair helicasemore » activity and likely affect HD2 functional movement. TTD mutants lose or retain helicase activity but map to sites in all four domains expected to cause framework defects impacting TFIIH integrity. These results provide a foundation for understanding disease consequences of mutations in XPD and related 4Fe-4S helicases including FancJ.« less
-
DOE Office of Scientific and Technical Information (OSTI.GOV)
Tainer, John; Fan, Li; Fuss, Jill O.
2008-06-02
Mutations in XPD helicase, required for nucleotide excision repair (NER) as part of the transcription/repair complex TFIIH, cause three distinct phenotypes: cancer-prone xeroderma pigmentosum (XP), or aging disorders Cockayne syndrome (CS), and trichothiodystrophy (TTD). To clarify molecular differences underlying these diseases, we determined crystal structures of the XPD catalytic core from Sulfolobus acidocaldarius and measured mutant enzyme activities. Substrate-binding grooves separate adjacent Rad51/RecA-like helicase domains (HD1, HD2) and an arch formed by 4FeS and Arch domains. XP mutations map along the HD1 ATP-binding edge and HD2 DNA-binding channel and impair helicase activity essential for NER. XP/CS mutations both impair helicasemore » activity and likely affect HD2 functional movement. TTD mutants lose or retain helicase activity but map to sites in all four domains expected to cause framework defects impacting TFIIH integrity. These results provide a foundation for understanding disease consequences of mutations in XPD and related 4Fe-4S helicases including FancJ.« less
-
Viral peptides-MHC interaction: Binding probability and distance from human peptides.
Santoni, Daniele
2018-05-23
Identification of peptides binding to MHC class I complex can play a crucial role in retrieving potential targets able to trigger an immune response. Affinity binding of viral peptides can be estimated through effective computational methods that in the most of cases are based on machine learning approach. Achieving a better insight into peptide features that impact on the affinity binding rate is a challenging issue. In the present work we focused on 9-mer peptides of Human immunodeficiency virus type 1 and Human herpes simplex virus 1, studying their binding to MHC class I. Viral 9-mers were partitioned into different classes, where each class is characterized by how far (in terms of mutation steps) the peptides belonging to that class are from human 9-mers. Viral 9-mers were partitioned in different classes, based on the number of mutation steps they are far from human 9-mers. We showed that the overall binding probability significantly differs among classes, and it typically increases as the distance, computed in terms of number of mutation steps from the human set of 9-mers, increases. The binding probability is particularly high when considering viral 9-mers that are far from all human 9-mers more than three mutation steps. A further evidence, providing significance to those special viral peptides and suggesting a potential role they can play, comes from the analysis of their distribution along viral genomes, as it revealed they are not randomly located, but they preferentially occur in specific genes. Copyright © 2018 Elsevier B.V. All rights reserved.
-
Delineation of the Marfan phenotype associated with mutations in exons 23-32 of the FBN1 gene
DOE Office of Scientific and Technical Information (OSTI.GOV)
Putnam, E.A.; Cho, M.; Milewicz, D.M.
Marfan syndrome is a dominantly inherited connective tissue disorder with a wide range of phenotypic severity. The condition is the result of mutations in FBN1, a large gene composed of 65 exons encoding the fibrillin-1 protein. While mutations causing classic manifestations of Marfan syndrome have been identified throughout the FBN1 gene, the six previously characterized mutations resulting in the severe, perinatal lethal form of Marfan syndrome have clustered in exons 24-32 of the gene. We screened 8 patients with either neonatal Marfan syndrome or severe cardiovascular complications of Marfan syndrome for mutations in this region of the gene. Using intron-basedmore » exon-specific primers, we amplified exons 23-32 from genomic DNAs, screened these fragments by single-stranded conformational polymorphism analysis, and sequenced indicated exons. This analysis documented mutations in exons 25-27 of the FBN1 mutations in 6 of these patients. These results, taken together with previously published FBN1 mutations in this region, further define the phenotype associated with mutations in exons 24-32 of the FBN1 gene, information important for the development of possible diagnostic tests and genetic counseling. 49 refs., 4 figs., 2 tabs.« less
-
Wood-Bouwens, Christina; Lau, Billy T; Handy, Christine M; Lee, HoJoon; Ji, Hanlee P
2017-09-01
We describe a single-color digital PCR assay that detects and quantifies cancer mutations directly from circulating DNA collected from the plasma of cancer patients. This approach relies on a double-stranded DNA intercalator dye and paired allele-specific DNA primer sets to determine an absolute count of both the mutation and wild-type-bearing DNA molecules present in the sample. The cell-free DNA assay uses an input of 1 ng of nonamplified DNA, approximately 300 genome equivalents, and has a molecular limit of detection of three mutation DNA genome-equivalent molecules per assay reaction. When using more genome equivalents as input, we demonstrated a sensitivity of 0.10% for detecting the BRAF V600E and KRAS G12D mutations. We developed several mutation assays specific to the cancer driver mutations of patients' tumors and detected these same mutations directly from the nonamplified, circulating cell-free DNA. This rapid and high-performance digital PCR assay can be configured to detect specific cancer mutations unique to an individual cancer, making it a potentially valuable method for patient-specific longitudinal monitoring. Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.
-
Moscardini, Mila; Pistello, Mauro; Bendinelli, M; Ficheux, Damien; Miller, Jennifer T; Gabus, Caroline; Le Grice, Stuart F J; Surewicz, Witold K; Darlix, Jean-Luc
2002-04-19
All lentiviruses and oncoretroviruses examined so far encode a major nucleic-acid binding protein (nucleocapsid or NC* protein), approximately 2500 molecules of which coat the dimeric RNA genome. Studies on HIV-1 and MoMuLV using in vitro model systems and in vivo have shown that NC protein is required to chaperone viral RNA dimerization and packaging during virus assembly, and proviral DNA synthesis by reverse transcriptase (RT) during infection. The human cellular prion protein (PrP), thought to be the major component of the agent causing transmissible spongiform encephalopathies (TSE), was recently found to possess a strong affinity for nucleic acids and to exhibit chaperone properties very similar to HIV-1 NC protein in the HIV-1 context in vitro. Tight binding of PrP to nucleic acids is proposed to participate directly in the prion disease process. To extend our understanding of lentiviruses and of the unexpected nucleic acid chaperone properties of the human prion protein, we set up an in vitro system to investigate replication of the feline immunodeficiency virus (FIV), which is functionally and phylogenetically distant from HIV-1. The results show that in the FIV model system, NC protein chaperones viral RNA dimerization, primer tRNA(Lys,3) annealing to the genomic primer-binding site (PBS) and minus strand DNA synthesis by the homologous FIV RT. FIV NC protein is able to trigger specific viral DNA synthesis by inhibiting self-priming of reverse transcription. The human prion protein was found to mimic the properties of FIV NC with respect to primer tRNA annealing to the viral RNA and chaperoning minus strand DNA synthesis. Copyright 2002 Elsevier Science Ltd.
-
Trbusek, Martin; Smardova, Jana; Malcikova, Jitka; Sebejova, Ludmila; Dobes, Petr; Svitakova, Miluse; Vranova, Vladimira; Mraz, Marek; Francova, Hana Skuhrova; Doubek, Michael; Brychtova, Yvona; Kuglik, Petr; Pospisilova, Sarka; Mayer, Jiri
2011-07-01
There is a distinct connection between TP53 defects and poor prognosis in chronic lymphocytic leukemia (CLL). It remains unclear whether patients harboring TP53 mutations represent a homogenous prognostic group. We evaluated the survival of patients with CLL and p53 defects identified at our institution by p53 yeast functional assay and complementary interphase fluorescence in situ hybridization analysis detecting del(17p) from 2003 to 2010. A defect of the TP53 gene was identified in 100 of 550 patients. p53 mutations were strongly associated with the deletion of 17p and the unmutated IgVH locus (both P < .001). Survival assessed from the time of abnormality detection was significantly reduced in patients with both missense (P < .001) and nonmissense p53 mutations (P = .004). In addition, patients harboring missense mutation located in p53 DNA-binding motifs (DBMs), structurally well-defined parts of the DNA-binding domain, manifested a clearly shorter median survival (12 months) compared with patients having missense mutations outside DBMs (41 months; P = .002) or nonmissense alterations (36 months; P = .005). The difference in survival was similar in the analysis limited to patients harboring mutation accompanied by del(17p) and was also confirmed in a subgroup harboring TP53 defect at diagnosis. The patients with p53 DBMs mutation (at diagnosis) also manifested a short median time to first therapy (TTFT; 1 month). The substantially worse survival and the short TTFT suggest a strong mutated p53 gain-of-function phenotype in patients with CLL with DBMs mutations. The impact of p53 DBMs mutations on prognosis and response to therapy should be analyzed in investigative clinical trials.
-
Structure of Hepatitis C Virus Polymerase in Complex with Primer-Template RNA
Murakami, Eisuke; Lam, Angela M.; Grice, Rena L.; Du, Jinfa; Sofia, Michael J.; Furman, Philip A.; Otto, Michael J.
2012-01-01
The replication of the hepatitis C viral (HCV) genome is accomplished by the NS5B RNA-dependent RNA polymerase (RdRp), for which mechanistic understanding and structure-guided drug design efforts have been hampered by its propensity to crystallize in a closed, polymerization-incompetent state. The removal of an autoinhibitory β-hairpin loop from genotype 2a HCV NS5B increases de novo RNA synthesis by >100-fold, promotes RNA binding, and facilitated the determination of the first crystallographic structures of HCV polymerase in complex with RNA primer-template pairs. These crystal structures demonstrate the structural realignment required for primer-template recognition and elongation, provide new insights into HCV RNA synthesis at the molecular level, and may prove useful in the structure-based design of novel antiviral compounds. Additionally, our approach for obtaining the RNA primer-template-bound structure of HCV polymerase may be generally applicable to solving RNA-bound complexes for other viral RdRps that contain similar regulatory β-hairpin loops, including bovine viral diarrhea virus, dengue virus, and West Nile virus. PMID:22496223
-
Joutel, Anne; Monet, Marie; Domenga, Valérie; Riant, Florence; Tournier-Lasserve, Elisabeth
2004-01-01
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited vascular dementia characterized by the degeneration of smooth-muscle cells in small cerebral arteries. CADASIL is caused by mutations in NOTCH3, one of the four mammalian homologs to the Drosophila melanogaster NOTCH gene. Disease-associated mutations are distributed throughout the 34 epidermal growth factor–like repeats (EGFRs) that compose the extracellular domain of the Notch3 receptor and result in a loss or a gain of a cysteine residue in one of these EGFRs. In human adults, Notch3 expression is highly restricted to vascular smooth-muscle cells. In patients with CADASIL, there is an abnormal accumulation of Notch3 in the vessel. Molecular pathways linking NOTCH3 mutations to degeneration of vascular smooth-muscle cells are as yet poorly understood. In this study, we investigated the effect of CADASIL mutations on Notch3 activity. We studied five naturally occurring mutations: R90C and C212S, located in the previously identified mutational hotspot EGFR2–5; C428S, shown in this study to be located in the ligand-binding domain EGFR10–11; and C542Y and R1006C, located in EGFR13 and EGFR26, respectively. All five mutant proteins were correctly processed. The C428S and C542Y mutant receptors exhibited a significant reduction in Jagged1-induced transcriptional activity of a RBP/JK responsive luciferase reporter, relative to wild-type Notch3. Impaired signaling activity of these two mutants arose through different mechanisms; the C428S mutant lost its Jagged1-binding ability, whereas C542Y retained it but exhibited an impaired presentation to the cell surface. In contrast, the R90C, C212S, and R1006C mutants retained the ability to bind Jagged1 and were associated with apparently normal levels of signaling activity. We conclude that mutations in Notch3 differently affect Jagged1 binding and Notch3 signaling via the RBP/JK pathway. PMID:14714274
-
Deng, Yi-Mo; Spirason, Natalie; Iannello, Pina; Jelley, Lauren; Lau, Hilda; Barr, Ian G
2015-07-01
Full genome sequencing of influenza A viruses (IAV), including those that arise from annual influenza epidemics, is undertaken to determine if reassorting has occurred or if other pathogenic traits are present. Traditionally IAV sequencing has been biased toward the major surface glycoproteins haemagglutinin and neuraminidase, while the internal genes are often ignored. Despite the development of next generation sequencing (NGS), many laboratories are still reliant on conventional Sanger sequencing to sequence IAV. To develop a minimal and robust set of primers for Sanger sequencing of the full genome of IAV currently circulating in humans. A set of 13 primer pairs was designed that enabled amplification of the six internal genes of multiple human IAV subtypes including the recent avian influenza A(H7N9) virus from China. Specific primers were designed to amplify the HA and NA genes of each IAV subtype of interest. Each of the primers also incorporated a binding site at its 5'-end for either a forward or reverse M13 primer, such that only two M13 primers were required for all subsequent sequencing reactions. This minimal set of primers was suitable for sequencing the six internal genes of all currently circulating human seasonal influenza A subtypes as well as the avian A(H7N9) viruses that have infected humans in China. This streamlined Sanger sequencing protocol could be used to generate full genome sequence data more rapidly and easily than existing influenza genome sequencing protocols. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.
-
Hanning, Jennifer E; Groves, Ian J; Pett, Mark R; Coleman, Nicholas
2013-05-21
Short interfering RNAs (siRNAs) are often used to deplete viral polycistronic transcripts, such as those encoded by human papillomavirus (HPV). There are conflicting data in the literature concerning how siRNAs targeting one HPV gene can affect levels of other genes in the polycistronic transcripts. We hypothesised that the conflict might be partly explained by the method of cDNA synthesis used prior to transcript quantification. We treated HPV16-positive cervical keratinocytes with siRNAs targeting the HPV16 E7 gene and used quantitative PCR to compare transcript levels of E7 with those of E6 and E2, viral genes located upstream and downstream of the target site respectively. We compared our findings from cDNA generated using oligo-dT primers alone with those from cDNA generated using a combination of random hexamer and oligo-dT primers. Our data show that when polycistronic transcripts are targeted by siRNAs, there is a period when untranslatable cleaved mRNA upstream of the siRNA binding site remains detectable by PCR, if cDNA is generated using random hexamer primers. Such false indications of mRNA abundance are avoided using oligo-dT primers. The period corresponds to the time taken for siRNA activity and degradation of the cleaved transcripts. Genes downstream of the siRNA binding site are detectable during this interval, regardless of how the cDNA is generated. These data emphasise the importance of the cDNA synthesis method used when measuring transcript abundance following siRNA depletion of polycistronic transcripts. They provide a partial explanation for erroneous reports suggesting that siRNAs targeting HPV E7 can have gene-specific effects.
-
2013-01-01
Background Short interfering RNAs (siRNAs) are often used to deplete viral polycistronic transcripts, such as those encoded by human papillomavirus (HPV). There are conflicting data in the literature concerning how siRNAs targeting one HPV gene can affect levels of other genes in the polycistronic transcripts. We hypothesised that the conflict might be partly explained by the method of cDNA synthesis used prior to transcript quantification. Findings We treated HPV16-positive cervical keratinocytes with siRNAs targeting the HPV16 E7 gene and used quantitative PCR to compare transcript levels of E7 with those of E6 and E2, viral genes located upstream and downstream of the target site respectively. We compared our findings from cDNA generated using oligo-dT primers alone with those from cDNA generated using a combination of random hexamer and oligo-dT primers. Our data show that when polycistronic transcripts are targeted by siRNAs, there is a period when untranslatable cleaved mRNA upstream of the siRNA binding site remains detectable by PCR, if cDNA is generated using random hexamer primers. Such false indications of mRNA abundance are avoided using oligo-dT primers. The period corresponds to the time taken for siRNA activity and degradation of the cleaved transcripts. Genes downstream of the siRNA binding site are detectable during this interval, regardless of how the cDNA is generated. Conclusions These data emphasise the importance of the cDNA synthesis method used when measuring transcript abundance following siRNA depletion of polycistronic transcripts. They provide a partial explanation for erroneous reports suggesting that siRNAs targeting HPV E7 can have gene-specific effects. PMID:23693071
-
Fluorescence studies with DNA probes: dynamic aspects of DNA structure and DNA-protein interactions
NASA Astrophysics Data System (ADS)
Millar, David P.; Carver, Theodore E.
1994-08-01
Time-resolved fluorescence measurements of optical probes incorporated at specific sites in DNA provides a new approach to studies of DNA structure and DNA:protein interactions. This approach can be used to study complex multi-state behavior, such as the folding of DNA into alternative higher order structures or the transfer of DNA between multiple binding sites on a protein. In this study, fluorescence anisotropy decay of an internal dansyl probe attached to 17/27-mer oligonucleotides was used to monitor the distribution of DNA 3' termini bound at either the polymerase of 3' to 5' exonuclease sites of the Klenow fragment of DNA polymerase I. Partitioning of the primer terminus between the two active sites of the enzyme resulted in a heterogeneous probe environment, reflected in the associative behavior of the fluorescence anisotropy decay. Analysis of the anisotropy decay with a two state model of solvent-exposed and protein-associated dansyl probes was used to determine the fraction of DNA bound at each site. We examined complexes of Klenow fragment with DNAs containing various base mismatches. Single mismatches at the primer terminus caused a 3-fold increase in the equilibrium partitioning of DNA into the exonuclease site, while two or more consecutive G:G mismatches caused the DNA to bind exclusively at the exonuclease site, with a partitioning constant at least 250- fold greater than that of the corresponding matched DNA sequence. Internal single mismatches located up to four bases from the primer terminus produced larger effects than the same mismatch at the primer terminus. These results provide insight into the recognition mechanisms that enable DNA polymerases to proofread misincorporated bases during DNA replication.
-
Cross, F R; Levine, K
2000-01-01
We showed recently that a screen for mutant CDC28 with improved binding to a defective Cln2p G1 cyclin yielded a spectrum of mutations similar to those yielded by a screen for intragenic suppressors of the requirement for activation loop phosphorylation (T169E suppressors). Recombination among these mutations yielded CDC28 mutants that bypassed the G1 cyclin requirement. Here we analyze further the interrelationship between T169E suppression, interaction with defective cyclin, and G1 cyclin bypass. DNA shuffling of mutations from the various screens and recombination onto a T169E-encoding 3' end yielded CDC28 mutants with strong T169E suppression. Some of the strongest T169E suppressors could suppress the defective Cln2p G1 cyclin even while retaining T169E. The strong T169E suppressors did not exhibit bypass of the G1 cyclin requirement but did so when T169E was reverted to T. These results suggested that for these mutants, activation loop phosphorylation and cyclin binding might be alternative means of activation rather than independent requirements for activation (as with wild type). These results suggest mechanistic overlap between the conformational shift induced by cyclin binding and that induced by activation loop phosphorylation. This conclusion was supported by analysis of suppressors of a mutation in the Cdk phosphothreonine-binding pocket created by cyclin binding. PMID:10747052
-
Qiu, Yimin; Mekkat, Arya; Yu, Hongtao; Yigit, Sezin; Hamaia, Samir; Farndale, Richard W; Kaplan, David L; Lin, Yu-Shan; Brodsky, Barbara
2018-05-11
Gly missense mutations in type I collagen, which replace a conserved Gly in the repeating (Gly-Xaa-Yaa) n sequence with a larger residue, are known to cause Osteogenesis Imperfecta (OI). The clinical consequences of such mutations range from mild to lethal, with more serious clinical severity associated with larger Gly replacement residues. Here, we investigate the influence of the identity of the residue replacing Gly within and adjacent to the integrin binding 502 GFPGER 507 sequence on triple-helix structure, stability and integrin binding using a recombinant bacterial collagen system. Recombinant collagens were constructed with Gly substituted by Ala, Ser or Val at four positions within the integrin binding region. All constructs formed a stable triple-helix structure with a small decrease in melting temperature. Trypsin was used to probe local disruption of the triple helix, and Gly to Val replacements made the triple helix trypsin sensitive at three of the four sites. Any mutation at Gly505, eliminated integrin binding, while decreased integrin binding affinity was observed in the replacement of Gly residues at Gly502 following the order Val > Ser > Ala. Molecular dynamics simulations indicated that all Gly replacements led to transient disruption of triple-helix interchain hydrogen bonds in the region of the Gly replacement. These computational and experimental results lend insight into the complex molecular basis of the varying clinical severity of OI. Copyright © 2018. Published by Elsevier Inc.
-
Feng, Yu; Chen, Runsen; Da, Min; Qian, Bo; Mo, Xuming
2018-06-30
Esophageal atresia and tracheoesophageal fistula (EA/TEF) are relatively common malformations in newborns, but the etiology of EA/TEF remains unknown. Fanconi anemia (FA) complementation group A (FANCA) is a key component of the FA core complex and is essential for the activation of the DNA repair pathway. The middle region (amino acids 674-1208) of FANCA is required for its interaction with FAAP20. We performed targeted sequencing of this binding region of FANCA (exons 23-36) in 40 EA/TEF patients. We also investigated the effect of the p.A958V mutation on the protein-protein interaction between FANCA and FAAP20 using an in vitro binding assay and co-immunoprecipitation. Immunolocalization analysis was performed to investigate the subcellular localization of FANCA, and tissue sections and immunohistochemistry were used to explore the expression of FANCA. We identified four rare missense variants in the FANCA binding region. FANCA mutations were significantly overrepresented in EA/TEF patients compared with 4300 control subjects from the NHLBI-ESP project (Fisher's exact p = 2.17 × 10 -5 , odds ratio = 31.75). p.A958V, a novel de novo mutation in the FANCA gene, was identified in one patient with EA/TEF. We provide further evidence that the p.A958V mutation reduces the binding affinity of FANCA for FAAP20. Interestingly, the p.A958V mutation impaired the nuclear localization of the FANCA protein expressed in HeLa cells. We found that FANCA was more highly expressed in stratified squamous epithelium than in smooth muscle. In conclusion, mutations in the FANCA gene are associated with EA/TEF in humans. Copyright © 2018. Published by Elsevier B.V.
-
Mutations in the putative calcium-binding domain of polyomavirus VP1 affect capsid assembly
NASA Technical Reports Server (NTRS)
Haynes, J. I. 2nd; Chang, D.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)
1993-01-01
Calcium ions appear to play a major role in maintaining the structural integrity of the polyomavirus and are likely involved in the processes of viral uncoating and assembly. Previous studies demonstrated that a VP1 fragment extending from Pro-232 to Asp-364 has calcium-binding capabilities. This fragment contains an amino acid stretch from Asp-266 to Glu-277 which is quite similar in sequence to the amino acids that make up the calcium-binding EF hand structures found in many proteins. To assess the contribution of this domain to polyomavirus structural integrity, the effects of mutations in this region were examined by transfecting mutated viral DNA into susceptible cells. Immunofluorescence studies indicated that although viral protein synthesis occurred normally, infective viral progeny were not produced in cells transfected with polyomavirus genomes encoding either a VP1 molecule lacking amino acids Thr-262 through Gly-276 or a VP1 molecule containing a mutation of Asp-266 to Ala. VP1 molecules containing the deletion mutation were unable to bind 45Ca in an in vitro assay. Upon expression in Escherichia coli and purification by immunoaffinity chromatography, wild-type VP1 was isolated as pentameric, capsomere-like structures which could be induced to form capsid-like structures upon addition of CaCl2, consistent with previous studies. However, although VP1 containing the point mutation was isolated as pentamers which were indistinguishable from wild-type VP1 pentamers, addition of CaCl2 did not result in their assembly into capsid-like structures. Immunogold labeling and electron microscopy studies of transfected mammalian cells provided in vivo evidence that a mutation in this region affects the process of viral assembly.
-
Srikumar, P S; Rohini, K; Rajesh, Perumbilavil Kaithamanakallam
2014-06-01
Mutations in human laforin lead to an autosomal neurodegenerative disorder Lafora disease. In N-terminal carbohydrate binding domain of laforin, two mutations W32G and K87A are reported as highly disease causing laforin mutants. Experimental studies reported that mutations are responsible for the abolishment of glycogen binding which is a critical function of laforin. Our current computational study focused on the role of conformational changes in human laforin structure due to existing single mutation W32G and prepared double mutation W32G/K87A related to loss of glycogen binding. We performed 10 ns molecular dynamics (MD) simulation studies in the Gromacs package for both mutations and analyzed the trajectories. From the results, the global properties like root mean square deviation, root mean square fluctuation, radius of gyration, solvent accessible surface area and hydrogen bonds showed structural changes in atomic level observed in W32G and W32G/K87A laforin mutants. The conformational change induced by mutants influenced the loss of the overall stability of the native laforin. Moreover, the change in overall motion of protein was analyzed by principal component analysis and results showed protein clusters expanded more than native and also change in direction in case of double mutant in conformational space. Overall, our report provides theoretical information on loss of structure-function relationship due to flexible nature of laforin mutants. In conclusion, comparative MD simulation studies support the experimental data on W32G and W32G/K87A related to the lafora disease mechanism on glycogen binding.
-
Structural basis of Bloom syndrome (BS) causing mutations in the BLM helicase domain.
Rong, S. B.; Väliaho, J.; Vihinen, M.
2000-01-01
BACKGROUND: Bloom syndrome (BS) is characterized by mutations within the BLM gene. The Bloom syndrome protein (BLM) has similarity to the RecQ subfamily of DNA helicases, which contain seven conserved helicase domains and share significant sequence and structural similarity with the Rep and PcrA DNA helicases. We modeled the three-dimensional structure of the BLM helicase domain to analyze the structural basis of BS-causing mutations. MATERIALS AND METHODS: The sequence alignment was performed for RecQ DNA helicases and Rep and PcrA helicases. The crystal structure of PcrA helicase (PDB entry 3PJR) was used as the template for modeling the BLM helicase domain. The model was used to infer the function of BLM and to analyze the effect of the mutations. RESULTS: The structural model with good stereochemistry of the BLM helicase domain contains two subdomains, 1A and 2A. The electrostatic potential of the model is highly negative over most of the surface, except for the cleft between subdomains 1A and 2A which is similar to the template protein. The ATP-binding site is located inside the model between subdomains 1A and 2A; whereas, the DNA-binding region is situated at the surface cleft, with positive potential between 1A and 2A. CONCLUSIONS: The three-dimensional structure of the BLM helicase domain was modeled and applied to interpret BS-causing mutations. The mutation I841T is likely to weaken DNA binding, while the mutations C891R, C901Y, and Q672R presumably disturb the ATP binding. In addition, other critical positions are discussed. PMID:10965492
-
MLH1 mutations differentially affect meiotic functions in Saccharomyces cerevisiae.
Hoffmann, Eva R; Shcherbakova, Polina V; Kunkel, Thomas A; Borts, Rhona H
2003-01-01
To test whether missense mutations in the cancer susceptibility gene MLH1 adversely affect meiosis, we examined 14 yeast MLH1 mutations for effects on meiotic DNA transactions and gamete viability in the yeast Saccharomyces cerevisiae. Mutations analogous to those associated with hereditary nonpolyposis colorectal cancer (HNPCC) or those that reduce Mlh1p interactions with ATP or DNA all impair replicative mismatch repair as measured by increased mutation rates. However, their effects on meiotic heteroduplex repair, crossing over, chromosome segregation, and gametogenesis vary from complete loss of meiotic functions to no meiotic defect, and mutants defective in one meiotic process are not necessarily defective in others. DNA binding and ATP binding but not ATP hydrolysis are required for meiotic crossing over. The results reveal clear separation of different Mlh1p functions in mitosis and meiosis, and they suggest that some, but not all, MLH1 mutations may be a source of human infertility. PMID:12618391
-
Brief Report:MECP2 Mutations in People without Rett Syndrome
ERIC Educational Resources Information Center
Suter, Bernhard; Treadwell-Deering, Diane; Zoghbi, Huda Y.; Glaze, Daniel G.; Neul, Jeffrey L.
2014-01-01
Mutations in "Methyl-CpG-Binding protein 2" ("MECP2") are commonly associated with the neurodevelopmental disorder Rett syndrome (RTT). However, some people with RTT do not have mutations in "MECP2," and interestingly there have been people identified with "MECP2" mutations that do not have the clinical…
-
Harteveld, C L
2014-02-01
For detecting carriers of thalassemia traits, the basic part of diagnostics consists of measurement of the hematological indices followed by mostly automatic separation and measurement of the Hb fractions, while direct Hb separation either on high pressure liquid chromatography or capillary electrophoresis is sufficient to putatively identify carriers of the common Hb variants like HbS, C, E, D, and O-Arab. A putative positive result is reported together with an advice for parents, partner, or family analysis. For couples, presumed at-risk confirmation at the DNA level is essential. In general, this part of diagnostics is done in specialized centers provided with sufficient experience and the technical tools needed to combine hematological and biochemical interpretation with identification of the mutations at the molecular level. State-of-the-art tools are usually available in centers that also provide prenatal diagnosis and should consist of gap-PCR for the common deletions, direct DNA sequencing for all kind of point-mutations and the capacity to uncover novel or rare mutations or disease mechanisms. New developments are MLPA for large and eventually unknown deletion defects and microarray technology for fine mapping and primer design for breakpoint analysis. Gap-PCR primers designed in the region flanking the deletion breakpoints can subsequently be used to facilitate carrier detection of uncommon deletions in family members or isolated populations in laboratories where no microarray technology or MLPA is available. © 2013 John Wiley & Sons Ltd.
-
Saccharomyces cerevisiae Msh2-Msh3 acts in repair of base-base mispairs.
Harrington, Jill M; Kolodner, Richard D
2007-09-01
DNA mismatch repair is thought to act through two subpathways involving the recognition of base-base and insertion/deletion mispairs by the Msh2-Msh6 heterodimer and the recognition of insertion/deletion mispairs by the Msh2-Msh3 heterodimer. Here, through genetic and biochemical approaches, we describe a previously unidentified role of the Msh2-Msh3 heterodimer in the recognition of base-base mispairs and the suppression of homology-mediated duplication and deletion mutations. Saccharomyces cerevisiae msh3 mutants did not show an increase in the rate of base substitution mutations by the CAN1 forward mutation assay compared to the rate for the wild type but did show an altered spectrum of base substitution mutations, including an increased accumulation of base pair changes from GC to CG and from AT to TA; msh3 mutants also accumulated homology-mediated duplication and deletion mutations. The mutation spectrum of mlh3 mutants paralleled that of msh3 mutants, suggesting that the Mlh1-Mlh3 heterodimer may also play a role in the repair of base-base mispairs and in the suppression of homology-mediated duplication and deletion mutations. Mispair binding analysis with purified Msh2-Msh3 and DNA substrates derived from CAN1 sequences found to be mutated in vivo demonstrated that Msh2-Msh3 exhibited robust binding to specific base-base mispairs that was consistent with functional mispair binding.
-
Saccharomyces cerevisiae Msh2-Msh3 Acts in Repair of Base-Base Mispairs▿ †
Harrington, Jill M.; Kolodner, Richard D.
2007-01-01
DNA mismatch repair is thought to act through two subpathways involving the recognition of base-base and insertion/deletion mispairs by the Msh2-Msh6 heterodimer and the recognition of insertion/deletion mispairs by the Msh2-Msh3 heterodimer. Here, through genetic and biochemical approaches, we describe a previously unidentified role of the Msh2-Msh3 heterodimer in the recognition of base-base mispairs and the suppression of homology-mediated duplication and deletion mutations. Saccharomyces cerevisiae msh3 mutants did not show an increase in the rate of base substitution mutations by the CAN1 forward mutation assay compared to the rate for the wild type but did show an altered spectrum of base substitution mutations, including an increased accumulation of base pair changes from GC to CG and from AT to TA; msh3 mutants also accumulated homology-mediated duplication and deletion mutations. The mutation spectrum of mlh3 mutants paralleled that of msh3 mutants, suggesting that the Mlh1-Mlh3 heterodimer may also play a role in the repair of base-base mispairs and in the suppression of homology-mediated duplication and deletion mutations. Mispair binding analysis with purified Msh2-Msh3 and DNA substrates derived from CAN1 sequences found to be mutated in vivo demonstrated that Msh2-Msh3 exhibited robust binding to specific base-base mispairs that was consistent with functional mispair binding. PMID:17636021
-
Yang, Bei; Zhang, Haiping; Wang, Hao
2015-02-01
HER2, a member of the human ErbB protein family belonging to receptor tyrosine kinases, plays important roles in regulating crucial cellular processes, including cell migration, proliferation, and differentiation. A missense mutation, L755P, in the HER2 kinase domain has been involved in lung cancer in humans and exhibits reduced response to lapatinib therapy. However, the detailed mechanism of how the HER2 L755P mutation causes drug resistance to lapatinib remains elusive. Here, molecular docking, molecular dynamics (MD) simulations, binding free energy calculations [molecular mechanics and generalized Born/surface area (MM-GBSA)] were performed to reveal the mechanism of drug resistance due to the HER2 L755P mutation. MD simulations revealed that the L755P mutation caused structural changes in the regions of helix αC, the glycine-rich loop, and the activation loop, thereby leading to the loss of interactions between the solubilizing group of lapatinib and HER2. Moreover, MM-GBSA calculations suggested that hydrophobic interactions between lapatinib and HER2 contribute most to the binding affinity, and that the L755P mutation could result in a less energetically favorable HER2/lapatinib complex. This may weaken the binding of lapatinib to the mutated HER2, thereby leading to the emergence of drug resistance. This study offers a structural explanation for the effect of the L755P mutation on the HER2/lapatinib complex.
-
Impact of mutations on the allosteric conformational equilibrium
Weinkam, Patrick; Chen, Yao Chi; Pons, Jaume; Sali, Andrej
2012-01-01
Allostery in a protein involves effector binding at an allosteric site that changes the structure and/or dynamics at a distant, functional site. In addition to the chemical equilibrium of ligand binding, allostery involves a conformational equilibrium between one protein substate that binds the effector and a second substate that less strongly binds the effector. We run molecular dynamics simulations using simple, smooth energy landscapes to sample specific ligand-induced conformational transitions, as defined by the effector-bound and unbound protein structures. These simulations can be performed using our web server: http://salilab.org/allosmod/. We then develop a set of features to analyze the simulations and capture the relevant thermodynamic properties of the allosteric conformational equilibrium. These features are based on molecular mechanics energy functions, stereochemical effects, and structural/dynamic coupling between sites. Using a machine-learning algorithm on a dataset of 10 proteins and 179 mutations, we predict both the magnitude and sign of the allosteric conformational equilibrium shift by the mutation; the impact of a large identifiable fraction of the mutations can be predicted with an average unsigned error of 1 kBT. With similar accuracy, we predict the mutation effects for an 11th protein that was omitted from the initial training and testing of the machine-learning algorithm. We also assess which calculated thermodynamic properties contribute most to the accuracy of the prediction. PMID:23228330
-
Ridge, Justin P; Lin, Marianne; Larsen, Eloise I; Fegan, Mark; McEwan, Alastair G; Sly, Lindsay I
2007-04-01
Pedomicrobium sp. ACM 3067 is a budding-hyphal bacterium belonging to the alpha-Proteobacteria which is able to oxidize soluble Mn2+ to insoluble manganese oxide. A cosmid, from a whole-genome library, containing the putative genes responsible for manganese oxidation was identified and a primer-walking approach yielded 4350 bp of novel sequence. Analysis of this sequence showed the presence of a predicted three-gene operon, moxCBA. The moxA gene product showed homology to multicopper oxidases (MCOs) and contained the characteristic four copper-binding motifs (A, B, C and D) common to MCOs. An insertion mutation of moxA showed that this gene was essential for both manganese oxidation and laccase-like activity. The moxB gene product showed homology to a family of outer membrane proteins which are essential for Type I secretion in Gram-negative bacteria. moxBA has not been observed in other manganese-oxidizing bacteria but homologues were identified in the genomes of several bacteria including Sinorhizobium meliloti 1021 and Agrobacterium tumefaciens C58. These results suggest that moxBA and its homologues constitute a family of genes encoding an MCO and a predicted component of the Type I secretion system.
-
Huel, René L. M.; Bašić, Lara; Madacki-Todorović, Kamelija; Smajlović, Lejla; Eminović, Izet; Berbić, Irfan; Miloš, Ana; Parsons, Thomas J.
2007-01-01
Aim To present a compendium of off-ladder alleles and other genotyping irregularities relating to rare/unexpected population genetic variation, observed in a large short tandem repeat (STR) database from Bosnia and Serbia. Methods DNA was extracted from blood stain cards relating to reference samples from a population of 32 800 individuals from Bosnia and Serbia, and typed using Promega’s PowerPlex®16 STR kit. Results There were 31 distinct off-ladder alleles were observed in 10 of the 15 STR loci amplified from the PowerPlex®16 STR kit. Of these 31 alleles, 3 have not been previously reported. Furthermore, 16 instances of triallelic patterns were observed in 9 of the 15 loci. Primer binding site mismatches that affected amplification were observed in two loci, D5S818 and D8S1179. Conclusion Instances of deviations from manufacturer’s allelic ladders should be expected and caution taken to properly designate the correct alleles in large DNA databases. Particular care should be taken in kinship matching or paternity cases as incorrect designation of any of these deviations from allelic ladders could lead to false exclusions. PMID:17696304
-
Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn's disease.
Mao, Liming; Kitani, Atsushi; Similuk, Morgan; Oler, Andrew J; Albenberg, Lindsey; Kelsen, Judith; Aktay, Atiye; Quezado, Martha; Yao, Michael; Montgomery-Recht, Kim; Fuss, Ivan J; Strober, Warren
2018-05-01
In these studies, we evaluated the contribution of the NLRP3 inflammasome to Crohn's disease (CD) in a kindred containing individuals having a missense mutation in CARD8, a protein known to inhibit this inflammasome. Whole exome sequencing and PCR studies identified the affected individuals as having a V44I mutation in a single allele of the T60 isoform of CARD8. The serum levels of IL-1β in the affected individuals were increased compared with those in healthy controls, and their peripheral monocytes produced increased amounts of IL-1β when stimulated by NLRP3 activators. Immunoblot studies probing the basis of these findings showed that mutated T60 CARD8 failed to downregulate the NLRP3 inflammasome because it did not bind to NLRP3 and inhibit its oligomerization. In addition, these studies showed that mutated T60 CARD8 exerted a dominant-negative effect by its capacity to bind to and form oligomers with unmutated T60 or T48 CARD8 that impeded their binding to NLRP3. Finally, inflammasome activation studies revealed that intact but not mutated CARD8 prevented NLRP3 deubiquitination and serine dephosphorylation. CD due to a CARD8 mutation was not effectively treated by anti-TNF-α, but did respond to IL-1β inhibitors. Thus, patients with anti-TNF-α-resistant CD may respond to this treatment option.
-
Liang, Yang; Tebaldi, Toma; Rejeski, Kai; Joshi, Poorval; Stefani, Giovanni; Taylor, Ashley; Song, Yuanbin; Vasic, Radovan; Maziarz, Jamie; Balasubramanian, Kunthavai; Ardasheva, Anastasia; Ding, Alicia; Quattrone, Alessandro; Halene, Stephanie
2018-06-01
Recurrent mutations in the splicing factor SRSF2 are associated with poor clinical outcomes in myelodysplastic syndromes (MDS). Their high frequency suggests these mutations drive oncogenesis, yet the molecular explanation for this process is unclear. SRSF2 mutations could directly affect pre-mRNA splicing of a vital gene product; alternatively, a whole network of gene products could be affected. Here we determine how SRSF2 mutations globally affect RNA binding and splicing in vivo using HITS-CLIP. Remarkably, the majority of differential binding events do not translate into alternative splicing of exons with SRSF2 P95H binding sites. Alternative splice alterations appear to be dominated by indirect effects. Importantly, SRSF2 P95H targets are enriched in RNA processing and splicing genes, including several members of the hnRNP and SR families of proteins, suggesting a "splicing-cascade" phenotype wherein mutation of a single splicing factor leads to widespread modifications in multiple RNA processing and splicing proteins. We show that splice alteration of HNRNPA2B1, a splicing factor differentially bound and spliced by SRSF2 P95H , impairs hematopoietic differentiation in vivo. Our data suggests a model whereby the recurrent mutations in splicing factors set off a cascade of gene regulatory events that together affect hematopoiesis and drive cancer.
-
Seong, Ki Moon; Park, Hweon; Kim, Seong Jung; Ha, Hyo Nam; Lee, Jae Yung; Kim, Joon
2007-06-01
A yeast transcriptional activator, Gcn4p, induces the expression of genes that are involved in amino acid and purine biosynthetic pathways under amino acid starvation. Gcn4p has an acidic activation domain in the central region and a bZIP domain in the C-terminus that is divided into the DNA-binding motif and dimerization leucine zipper motif. In order to identify amino acids in the DNA-binding motif of Gcn4p which are involved in transcriptional activation, we constructed mutant libraries in the DNA-binding motif through an innovative application of random mutagenesis. Mutant library made by oligonucleotides which were mutated randomly using the Poisson distribution showed that the actual mutation frequency was in good agreement with expected values. This method could save the time and effort to create a mutant library with a predictable mutation frequency. Based on the studies using the mutant libraries constructed by the new method, the specific residues of the DNA-binding domain in Gcn4p appear to be involved in the transcriptional activities on a conserved binding site.
-
Wen, Cheng; Ye, Anpei
2013-01-01
BRaf (B- Rapid Accelerated Fibrosarcoma) protein is an important serine/threonine-protein kinase. Two domains on BRaf can independently bind its upstream kinase, Ras (Rat Sarcoma) protein. These are the Ras binding domain (RBD) and cysteine-rich-domain (CRD). Herein we use customized optical tweezers to compare the Ras binding process in two pathological mutants of BRaf responsible for CFC syndrome, abbreviated BRaf (A246P) and BRaf (Q257R). The two mutants differ in their kinetics of Ras-binding, though both bind Ras with similar increased overall affinity. BRaf (A246P) exhibits a slightly higher Ras/CRD unbinding force and a significantly higher Ras/RBD unbinding force versus the wild type. The contrary phenomenon is observed in the Q257R mutation. Simulations of the unstressed-off rate, koff(0), yield results in accordance with the changes revealed by the mean unbinding force. Our approach can be applied to rapidly assess other mutated proteins to deduce the effects of mutation on their kinetics compared to wild type proteins and to each other. PMID:24409384
-
2009-01-01
Background One of the most common and efficient methods for detecting mutations in genes is PCR amplification followed by direct sequencing. Until recently, the process of designing PCR assays has been to focus on individual assay parameters rather than concentrating on matching conditions for a set of assays. Primers for each individual assay were selected based on location and sequence concerns. The two primer sequences were then iteratively adjusted to make the individual assays work properly. This generally resulted in groups of assays with different annealing temperatures that required the use of multiple thermal cyclers or multiple passes in a single thermal cycler making diagnostic testing time-consuming, laborious and expensive. These factors have severely hampered diagnostic testing services, leaving many families without an answer for the exact cause of a familial genetic disease. A search of GeneTests for sequencing analysis of the entire coding sequence for genes that are known to cause muscular dystrophies returns only a small list of laboratories that perform comprehensive gene panels. The hypothesis for the study was that a complete set of universal assays can be designed to amplify and sequence any gene or family of genes using computer aided design tools. If true, this would allow automation and optimization of the mutation detection process resulting in reduced cost and increased throughput. Results An automated process has been developed for the detection of deletions, duplications/insertions and point mutations in any gene or family of genes and has been applied to ten genes known to bear mutations that cause muscular dystrophy: DMD; CAV3; CAPN3; FKRP; TRIM32; LMNA; SGCA; SGCB; SGCG; SGCD. Using this process, mutations have been found in five DMD patients and four LGMD patients (one in the FKRP gene, one in the CAV3 gene, and two likely causative heterozygous pairs of variations in the CAPN3 gene of two other patients). Methods and assay sequences are reported in this paper. Conclusion This automated process allows laboratories to discover DNA variations in a short time and at low cost. PMID:19835634
-
Bennett, Richard R; Schneider, Hal E; Estrella, Elicia; Burgess, Stephanie; Cheng, Andrew S; Barrett, Caitlin; Lip, Va; Lai, Poh San; Shen, Yiping; Wu, Bai-Lin; Darras, Basil T; Beggs, Alan H; Kunkel, Louis M
2009-10-18
One of the most common and efficient methods for detecting mutations in genes is PCR amplification followed by direct sequencing. Until recently, the process of designing PCR assays has been to focus on individual assay parameters rather than concentrating on matching conditions for a set of assays. Primers for each individual assay were selected based on location and sequence concerns. The two primer sequences were then iteratively adjusted to make the individual assays work properly. This generally resulted in groups of assays with different annealing temperatures that required the use of multiple thermal cyclers or multiple passes in a single thermal cycler making diagnostic testing time-consuming, laborious and expensive.These factors have severely hampered diagnostic testing services, leaving many families without an answer for the exact cause of a familial genetic disease. A search of GeneTests for sequencing analysis of the entire coding sequence for genes that are known to cause muscular dystrophies returns only a small list of laboratories that perform comprehensive gene panels.The hypothesis for the study was that a complete set of universal assays can be designed to amplify and sequence any gene or family of genes using computer aided design tools. If true, this would allow automation and optimization of the mutation detection process resulting in reduced cost and increased throughput. An automated process has been developed for the detection of deletions, duplications/insertions and point mutations in any gene or family of genes and has been applied to ten genes known to bear mutations that cause muscular dystrophy: DMD; CAV3; CAPN3; FKRP; TRIM32; LMNA; SGCA; SGCB; SGCG; SGCD. Using this process, mutations have been found in five DMD patients and four LGMD patients (one in the FKRP gene, one in the CAV3 gene, and two likely causative heterozygous pairs of variations in the CAPN3 gene of two other patients). Methods and assay sequences are reported in this paper. This automated process allows laboratories to discover DNA variations in a short time and at low cost.
-
Mohite, Ganesh M; Kumar, Rakesh; Panigrahi, Rajlaxmi; Navalkar, Ambuja; Singh, Nitu; Datta, Debalina; Mehra, Surabhi; Ray, Soumik; Gadhe, Laxmikant G; Das, Subhadeep; Singh, Namrata; Chatterjee, Debdeep; Kumar, Ashutosh; Maji, Samir K
2018-05-17
The involvement of α-synuclein (α-Syn) amyloid formation in Parkinson's disease (PD) pathogenesis is supported by the discovery of α-Syn gene (SNCA) mutations linked with familial PD, which are known to modulate the oligomerization and aggregation of α-Syn. Recently, the A53V mutation has been discovered, which leads to the late-onset PD. In the present study, we characterized for the first time the biophysical properties including the aggregation propensities, toxicity of aggregated species and membrane binding capability of A53V along with all familial mutations at A53 position. Present data suggest that A53V accelerate fibrillation of α-Syn without affecting the overall morphology and cytotoxicity of fibrils compared to wild-type protein. The aggregation propensity for A53 mutants is found to be; A53T>A53V>WT>A53E. Further, time course aggregation study reveals that A53V mutant promotes early oligomerization similar to A53T mutation. It promotes the highest amount of oligomer formation immediate after dissolution, which are cytotoxic. Although in the presence of membrane-mimicking environments, A53V mutation showed similar extent of helix-induction capacity as of WT protein, however, it exhibited lesser binding to lipid vesicle. The NMR study revealed unique chemical shift perturbation by A53V mutation com-pared to other mutations at A53 site. The present study might help to establish the disease-causing mechanism of A53V in PD pathology.
-
Characterization of calmodulin binding domains in TRPV2 and TRPV5 C-tails.
Holakovska, Blanka; Grycova, Lenka; Bily, Jan; Teisinger, Jan
2011-02-01
The transient receptor potential channels TRPV2 and TRPV5 belong to the vanilloid TRP subfamily. TRPV2 is highly similar to TRPV1 and shares many common properties with it. TRPV5 (and also its homolog TRPV6) is a rather distinct member of the TRPV subfamily. It is distant for being strictly Ca(2+)-selective and features quite different properties from the rest of the TRPV subfamily. It is known that TRP channels are regulated by calmodulin in a calcium-dependent manner. In our study we identified a calmodulin binding site on the C-termini of TRPV2 (654-683) and TRPV5 (587-616) corresponding to the consensus CaM binding motif 1-5-10. The R679 and K681 single mutants of TRPV2 caused a 50% decrease in binding affinity and a double mutation of K661/K664 of the same peptide lowered the binding affinity by up to 75%. A double mutation of R606/K607 and triple mutation of R594/R606/R610 in TRPV5 C-terminal peptide resulted in the total loss of binding affinity to calmodulin. These results demonstrate that the TRPV2 C-tail and TRPV5 C-tail contain calmodulin binding sites and that the basic residues are strongly involved in TRP channel binding to calmodulin.
-
Chen, Xi; Dou, Hu; Wang, Xingjuan; Huang, Yi; Lu, Ling; Bin, Junqing; Su, Yongchun; Zou, Lin; Yu, Jie; Bao, Liming
2018-04-01
The prevalence and clinical relevance of KIT mutations in childhood core-binding factor (CBF) acute myeloid leukemia (AML) have not been well characterized. In this study, a total of 212 children with de novo AML were enrolled from a Chinese population and 50 (23.5%) of the patients were deemed CBF-AML. KIT mutations were identified in 30% of the CBF-AML cohort. The KIT mutations were clustered in exon 17 and exon 8, and KIT mutations in exons 8 and 17 were correlated with a shorter overall survival (OS) (5-year OS: 30.0 ± 14.5% vs. 73.0 ± 8.5%, p = .007) and event-free survival (EFS) (5-year EFS: 30.0 ± 14.5% vs. 73.0 ± 8.5%, p = .003). Multivariate analysis revealed KIT mutations as an independent risk factor in CBF-AML. Our results suggest that KIT mutations are a molecular marker for an inferior prognosis in pediatric CBF-AML.
-
Fox, Jerome M.; Kang, Kyungtae; Sastry, Madhavi; ...
2017-03-02
In this study we use mutants of human carbonic anhydrase (HCAII) to examine how changes in the organization of water within a binding pocket can alter the thermodynamics of protein–ligand association. Results from calorimetric, crystallographic, and theoretical analyses suggest that most mutations strengthen networks of water-mediated hydrogen bonds and reduce binding affinity by increasing the enthalpic cost and, to a lesser extent, the entropic benefit of rearranging those networks during binding. The organization of water within a binding pocket can thus determine whether the hydrophobic interactions in which it engages are enthalpy-driven or entropy-driven. Our findings highlight a possible asymmetrymore » in protein–ligand association by suggesting that, within the confines of the binding pocket of HCAII, binding events associated with enthalpically favorable rearrangements of water are stronger than those associated with entropically favorable ones.« less
-
DOE Office of Scientific and Technical Information (OSTI.GOV)
Agniswamy, Johnson; Louis, John M.; Roche, Julien
We report structural analysis of HIV protease variant PRS17 which was rationally selected by machine learning to represent wide classes of highly drug-resistant variants. Crystal structures were solved of PRS17 in the inhibitor-free form and in complex with antiviral inhibitor, darunavir. Despite its 17 mutations, PRS17 has only one mutation (V82S) in the inhibitor/substrate binding cavity, yet exhibits high resistance to all clinical inhibitors. PRS17 has none of the major mutations (I47V, I50V, I54ML, L76V and I84V) associated with darunavir resistance, but has 10,000-fold weaker binding affinity relative to the wild type PR. Comparable binding affinity of 8000-fold weaker thanmore » PR is seen for drug resistant mutant PR20, which bears 3 mutations associated with major resistance to darunavir (I47V, I54L and I84V). Inhibitor-free PRS17 shows an open flap conformation with a curled tip correlating with G48V flap mutation. NMR studies on inactive PRS17 D25N unambiguously confirm that the flaps adopt mainly an open conformation in solution very similar to that in the inhibitor-free crystal structure. In PRS17, the hinge loop cluster of mutations, E35D, M36I and S37D, contributes to the altered flap dynamics by a mechanism similar to that of PR20. An additional K20R mutation anchors an altered conformation of the hinge loop. Flap mutations M46L and G48V in PRS17/DRV complex alter the Phe53 conformation by steric hindrance between the side chains. Unlike the L10F mutation in PR20, L10I in PRS17 does not break the inter-subunit ion pair or diminish the dimer stability, consistent with a very low dimer dissociation constant comparable to that of wild type PR. Distal mutations A71V, L90M and I93L propagate alterations to the catalytic site of PRS17. PRS17 exhibits a molecular mechanism whereby mutations act synergistically to alter the flap dynamics resulting in significantly weaker binding yet maintaining active site contacts with darunavir.« less
-
Komori, S; Sakata, K; Kasumi, H; Tsuji, Y; Hamada, K; Koyama, K
1999-10-01
DNA analysis of the androgen receptor gene in a patient with complete androgen insensitivity syndrome identified a substitutional mutation (tyrosine converted to cysteine at position 571) in the DNA binding domain. In vitro transfection experiments with the patients' androgen receptor gene, indicated normal expression of the androgen receptor in transfected COS-7 cells compared to the wild type gene. There was also no evidence of impaired thermal stability of the 5 alpha-dihydrotestosterone-androgen receptor complex. However, the capacity of the androgen receptor to activate target gene transcription was found to be completely disrupted in a luciferase assay. These results confirmed that only one substitutional mutation in the DNA binding domain was related to the pathogenesis of the complete androgen insensitivity syndrome.
-
Characterization of phospholipase C gamma enzymes with gain-of-function mutations.
Everett, Katy L; Bunney, Tom D; Yoon, Youngdae; Rodrigues-Lima, Fernando; Harris, Richard; Driscoll, Paul C; Abe, Koichiro; Fuchs, Helmut; de Angelis, Martin Hrabé; Yu, Philipp; Cho, Wohnwa; Katan, Matilda
2009-08-21
Phospholipase C gamma isozymes (PLC gamma 1 and PLC gamma 2) have a crucial role in the regulation of a variety of cellular functions. Both enzymes have also been implicated in signaling events underlying aberrant cellular responses. Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we have recently identified single point mutations in murine PLC gamma 2 that lead to spontaneous inflammation and autoimmunity. Here we describe further, mechanistic characterization of two gain-of-function mutations, D993G and Y495C, designated as ALI5 and ALI14. The residue Asp-993, mutated in ALI5, is a conserved residue in the catalytic domain of PLC enzymes. Analysis of PLC gamma 1 and PLC gamma 2 with point mutations of this residue showed that removal of the negative charge enhanced PLC activity in response to EGF stimulation or activation by Rac. Measurements of PLC activity in vitro and analysis of membrane binding have suggested that ALI5-type mutations facilitate membrane interactions without compromising substrate binding and hydrolysis. The residue mutated in ALI14 (Tyr-495) is within the spPH domain. Replacement of this residue had no effect on folding of the domain and enhanced Rac activation of PLC gamma 2 without increasing Rac binding. Importantly, the activation of the ALI14-PLC gamma 2 and corresponding PLC gamma 1 variants was enhanced in response to EGF stimulation and bypassed the requirement for phosphorylation of critical tyrosine residues. ALI5- and ALI14-type mutations affected basal activity only slightly; however, their combination resulted in a constitutively active PLC. Based on these data, we suggest that each mutation could compromise auto-inhibition in the inactive PLC, facilitating the activation process; in addition, ALI5-type mutations could enhance membrane interaction in the activated state.
-
Augert, Arnaud; Zhang, Qing; Bates, Breanna; Cui, Min; Wang, Xiaofei; Wildey, Gary; Dowlati, Afshin; MacPherson, David
2017-01-01
Introduction SCLC is a lethal neuroendocrine tumor type that is highly prone to metastasis. There is an urgency to understand the mutated genes that promote SCLC, as there are no approved targeted therapies yet available. SCLC is rarely resected, limiting the number of samples available for genomic analyses of somatic mutations. Methods To identify potential driver mutations in human SCLC we sequenced the whole exomes of 18 primary SCLCs and seven cell lines along with matched normal controls. We extended these data by resequencing a panel of genes across 40 primary SCLCs and 48 cell lines. Results We report frequent mutations in the lysine methyltransferase 2D gene (KMT2D) (also known as MLL2), a key regulator of transcriptional enhancer function. KMT2D exhibited truncating nonsense/frameshift/splice site mutations in 8% of SCLC tumors and 17% of SCLC cell lines. We found that KMT2D mutation in human SCLC cell lines was associated with reduced lysine methyltransferase 2D protein levels and reduced monomethylation of histone H3 lysine 4, a mark associated with transcriptional enhancers. We also found mutations in other genes associated with transcriptional enhancer control, including CREB binding protein gene (CREBBP), E1A binding protein p300 gene (EP300), and chromodomain helicase DNA binding protein 7 gene (CHD7), and we report mutations in additional chromatin remodeling genes such as polybromo 1 gene (PBRM1). Conclusions These data indicate that KMT2D is one of the major mutated genes in SCLC, and they point to perturbation of transcriptional enhancer control as potentially contributing to SCLC. PMID:28007623
-
Jalili, Seifollah; Karami, Leila; Schofield, Jeremy
2013-06-01
Proline-rich homeodomain (PRH) is a regulatory protein controlling transcription and gene expression processes by binding to the specific sequence of DNA, especially to the sequence 5'-TAATNN-3'. The impact of base pair mutations on the binding between the PRH protein and DNA is investigated using molecular dynamics and free energy simulations to identify DNA sequences that form stable complexes with PRH. Three 20-ns molecular dynamics simulations (PRH-TAATTG, PRH-TAATTA and PRH-TAATGG complexes) in explicit solvent water were performed to investigate three complexes structurally. Structural analysis shows that the native TAATTG sequence forms a complex that is more stable than complexes with base pair mutations. It is also observed that upon mutation, the number and occupancy of the direct and water-mediated hydrogen bonds decrease. Free energy calculations performed with the thermodynamic integration method predict relative binding free energies of 0.64 and 2 kcal/mol for GC to AT and TA to GC mutations, respectively, suggesting that among the three DNA sequences, the PRH-TAATTG complex is more stable than the two mutated complexes. In addition, it is demonstrated that the stability of the PRH-TAATTA complex is greater than that of the PRH-TAATGG complex.
-
Herrera-Moyano, Emilia; Moriel-Carretero, María; Montelone, Beth A; Aguilera, Andrés
2014-12-01
The eukaryotic TFIIH complex is involved in Nucleotide Excision Repair and transcription initiation. We analyzed three yeast mutations of the Rad3/XPD helicase of TFIIH known as rem (recombination and mutation phenotypes). We found that, in these mutants, incomplete NER reactions lead to replication fork breaking and the subsequent engagement of the homologous recombination machinery to restore them. Nevertheless, the penetrance varies among mutants, giving rise to a phenotype gradient. Interestingly, the mutations analyzed reside at the ATP-binding groove of Rad3 and in vivo experiments reveal a gain of DNA affinity upon damage of the mutant Rad3 proteins. Since mutations at the ATP-binding groove of XPD in humans are present in the Xeroderma pigmentosum-Cockayne Syndrome (XP-CS), we recreated rem mutations in human cells, and found that these are XP-CS-like. We propose that the balance between the loss of helicase activity and the gain of DNA affinity controls the capacity of TFIIH to open DNA during NER, and its persistence at both DNA lesions and promoters. This conditions NER efficiency and transcription resumption after damage, which in human cells would explain the XP-CS phenotype, opening new perspectives to understand the molecular basis of the role of XPD in human disease.
-
Sang, Peng; Xie, Yue-Hui; Li, Lin-Hua; Ye, Yu-Jia; Hu, Wei; Wang, Jing; Wan, Wen; Li, Rui; Li, Long-Jun; Ma, Lin-Ling; Li, Zhi; Liu, Shu-Qun; Meng, Zhao-Hui
2017-04-01
Pyrroline-5-carboxylate reductase (P5CR), an enzyme with conserved housekeeping roles, is involved in the etiology of cutis laxa. While previous work has shown that the R119G point mutation in the P5CR protein is involved, the structural mechanism behind the pathology remains to be elucidated. In order to probe the role of the R119G mutation in cutis laxa, we performed molecular dynamics (MD) simulations, essential dynamics (ED) analysis, and Molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) binding free energy calculations on wild type (WT) and mutant P5CR-NAD complex. These MD simulations and ED analyses suggest that the R119G mutation decreases the flexibility of P5CR, specifically in the substrate binding pocket, which could decrease the kinetics of the cofactor entrance and egress. Furthermore, the MM-PBSA calculations suggest the R119G mutant has a lower cofactor binding affinity for NAD than WT. Our study provides insight into the possible role of the R119G mutation during interactions between P5CR and NAD, thus bettering our understanding of how the mutation promotes cutis laxa. Copyright © 2017 Elsevier Ltd. All rights reserved.
-
Effects of mutations at amino acid 61 in the arm of TF1 on its DNA-binding properties.
Sayre, M H; Geiduschek, E P
1990-12-20
Transcription factor 1 (TF1) is the Bacillus subtilis phage SPO1-encoded member of the family of bacterial DNA-binding proteins that includes Escherichia coli HU and integration host factor (IHF). We have initiated a mutational analysis of the TF1 molecule to understand better its unique DNA-binding properties and to investigate its physiological function. We report here the consequences of mutating the putative DNA-binding "arms" of TF1. At position 61 in its primary sequence, TF1 contains a Phe residue in place of the Arg residue found in all other known members of the HU family. Substituting polar, uncharged residues for Phe61 substantially reduced the DNA-binding affinity and site-selectivity of TF1 in vitro, whereas the substitution of Tyr had no effect. Substituting Trp or Arg for Phe61 had little effect on the affinity of TF1 for SPO1 DNA, but altered the electrophoretic mobilities of protein-DNA complexes in non-denaturing gels. The Arg61 substitution increased the affinity of the protein for non-specific sites on thymine-containing DNA, thus reducing the natural preference of TF1 for (5-hydroxymethyluracil)-containing DNA. The Phe61-to-Arg mutation was also correlated with decreased phage yield and aberrant regulation of viral protein synthesis in vivo.
-
Vieira, Marcos C; Zinder, Daniel; Cobey, Sarah
2018-01-01
Abstract High-affinity antibodies arise within weeks of infection from the evolution of B-cell receptors under selection to improve antigen recognition. This rapid adaptation is enabled by the distribution of highly mutable “hotspot” motifs in B-cell receptor genes. High mutability in antigen-binding regions (complementarity determining regions [CDRs]) creates variation in binding affinity, whereas low mutability in structurally important regions (framework regions [FRs]) may reduce the frequency of destabilizing mutations. During the response, loss of mutational hotspots and changes in their distribution across CDRs and FRs are predicted to compromise the adaptability of B-cell receptors, yet the contributions of different mechanisms to gains and losses of hotspots remain unclear. We reconstructed changes in anti-HIV B-cell receptor sequences and show that mutability losses were ∼56% more frequent than gains in both CDRs and FRs, with the higher relative mutability of CDRs maintained throughout the response. At least 21% of the total mutability loss was caused by synonymous mutations. However, nonsynonymous substitutions caused most (79%) of the mutability loss in CDRs. Because CDRs also show strong positive selection, this result suggests that selection for mutations that increase binding affinity contributed to loss of mutability in antigen-binding regions. Although recurrent adaptation to evolving viruses could indirectly select for high mutation rates, we found no evidence of indirect selection to increase or retain hotspots. Our results suggest mutability losses are intrinsic to both the neutral and adaptive evolution of B-cell populations and might constrain their adaptation to rapidly evolving pathogens such as HIV and influenza. PMID:29688540
-
Pepe, G; Vanegas, O C; Rickards, O; Giusti, B; Comeglio, P; Brunelli, T; Marcucci, R; Prisco, D; Gensini, G F; Abbate, R
1999-02-01
Mild hyperhomocysteinemia is associated to mutations either in cystathionine beta-synthase (CBS) or in 5,10-methylenetetrahydrofolate reductase (MTHFR) genes. In 1995, Sebastio et al. characterized a 68 bp insertion in cis with the most common CBS mutation (T833C) detected in homocystinuric patients. Recently, this double mutation has been detected in Italian and North-American controls. Compared to a group of patients affected by coronary artery disease, North-American controls showed not statistically significant difference. Moreover, Italian controls displayed a microheterogeneity in the mutant allele frequency distribution depending on their geographical origin (North or South of Italy). Aim of our study was to evaluate the prevalence of the double in cis mutation in different populations. We studied 377 healthy subjects belonging to various human groups. Genomic DNA, extracted from peripheral blood samples, was amplified using specific primers; PCR fragments were digested with Bsr I restriction enzyme to detect the double mutation. Our data show a significant heterogeneity among the populations studied, therefore this mutation turned out to be a reliable anthropogenetic marker. The distribution of the double mutation will contribute, with other DNA polymorphisms, to evaluate the genetic admixture of mixed populations such as Afro-Americans.
-
Tai, Chang-Long; Liu, Mei-Ying; Yu, Hsiao-Chi; Chiang, Chiang-Chuan; Chiang, Hung; Suen, Jeng-Hung; Kao, Shu-Min; Huang, Yu-Hsiu; Wu, Tina Jui-Ting; Yang, Chia-Feng; Tsai, Fang-Chih; Lin, Ching-Yuang; Chang, Jan-Gowth; Chen, Hong-Duo; Niu, Dau-Ming
2012-02-18
As an X-linked genetic disorder, Fabry disease was first thought to affect males only, and females were generally considered to be asymptomatic carriers. However, recent research suggests that female carriers of Fabry disease may still develop vital organ damage causing severe morbidity and mortality. In the previous newborn screening, from 299,007 newborns, we identified a total of 20 different Fabry mutations and 121 newborns with Fabry mutations. However, we found that most female carriers are not detected by enzyme assays. A streamlined method for high resolution melting (HRM) analysis was designed to screen for GLA gene mutations using a same PCR and melting program. Primer sets were designed to cover the 7 exons and the Chinese common intronic mutation, IVS4+919G>A of GLA gene. The HRM analysis was successful in identifying heterozygous and hemizygous patients with the 20 surveyed mutations. We were also successful in using this method to test dry blood spots of newborns afflicted with Fabry mutations without having to determine DNA concentration before PCR amplification. The results of this study show that HRM could be a reliable and sensitive method for use in the rapid screening of females for GLA mutations. Copyright © 2011 Elsevier B.V. All rights reserved.
-
Parra, Mario A; Sala, Sergio Della; Abrahams, Sharon; Logie, Robert H; Méndez, Luis Guillermo; Lopera, Francisco
2011-06-01
Short-term memory binding of visual features which are processed across different dimensions (shape-colour) is impaired in sporadic Alzheimer's disease, familial Alzheimer's disease, and in asymptomatic carriers of familial Alzheimer's disease. This study investigated whether Alzheimer's disease also impacts on within-dimension binding processes. The study specifically explored whether visual short-term memory binding of features of the same type (colour-colour) is sensitive to Alzheimer's disease. We used a neuropsychological battery and a short-term memory binding task to assess patients with sporadic Alzheimer's disease (Experiment 1), familial Alzheimer's disease (Experiment 2) due to the mutation E280A of the Presenilin-1 gene and asymptomatic carriers of the mutation. The binding task assessed change detection within arrays of unicoloured objects (Colour Only) or bicoloured objects the colours of which had to be remembered separately (Unbound Colours) or together (Bound Colours). Performance on the Bound Colours condition (1) explained the largest proportion of variance between patients (sporadic and familial Alzheimer's disease), (2) combined more sensitivity and specificity for the disease than other more traditional neuropsychological tasks, (3) identified asymptomatic carriers of the mutation even when traditional neuropsychological measures and other measures of short-term memory did not and, (4) contrary to shape-colour binding, correlated with measures of hippocampal functions. Colour-colour binding and shape-colour binding both appear to be sensitive to AD even though they seem to rely on different brain mechanisms. Copyright © 2011 Elsevier Ltd. All rights reserved.
-
Dansault, Anouk; David, Gabriel; Schwartz, Claire; Jaliffa, Carolina; Vieira, Véronique; de la Houssaye, Guillaume; Bigot, Karine; Catin, Françise; Tattu, Laurent; Chopin, Catherine; Halimi, Philippe; Roche, Olivier; Van Regemorter, Nicole; Munier, Francis; Schorderet, Daniel; Dufier, Jean-Louis; Marsac, Cécile; Ricquier, Daniel; Menasche, Maurice; Penfornis, Alfred; Abitbol, Marc
2007-04-02
The PAX6 gene was first described as a candidate for human aniridia. However, PAX6 expression is not restricted to the eye and it appears to be crucial for brain development. We studied PAX6 mutations in a large spectrum of patients who presented with aniridia phenotypes, Peters' anomaly, and anterior segment malformations associated or not with neurological anomalies. Patients and related families were ophthalmologically phenotyped, and in some cases neurologically and endocrinologically examined. We screened the PAX6 gene by direct sequencing in three groups of patients: those affected by aniridia; those with diverse ocular manifestations; and those with Peters' anomaly. Two mutations were investigated by generating crystallographic representations of the amino acid changes. Three novel heterozygous mutations affecting three unrelated families were identified: the g.572T>C nucleotide change, located in exon 5, and corresponding to the Leucine 46 Proline amino-acid mutation (L46P); the g.655A>G nucleotide change, located in exon 6, and corresponding to the Serine 74 Glycine amino-acid mutation (S74G); and the nucleotide deletion 579delG del, located in exon 6, which induces a frameshift mutation leading to a stop codon (V48fsX53). The L46P mutation was identified in affected patients presenting bilateral microphthalmia, cataracts, and nystagmus. The S74G mutation was found in a large family that had congenital ocular abnormalities, diverse neurological manifestations, and variable cognitive impairments. The 579delG deletion (V48fsX53) caused in the affected members of the same family bilateral aniridia associated with congenital cataract, foveal hypolasia, and nystagmus. We also detected a novel intronic nucleotide change, IVS2+9G>A (very likely a mutation) in an apparently isolated patient affected by a complex ocular phenotype, characterized primarily by a bilateral microphthalmia. Whether this nucleotide change is indeed pathogenic remains to be demonstrated. Two previously known heterozygous mutations of the PAX6 gene sequence were also detected in patients affected by aniridia: a de novo previously known nucleotide change, g.972C>T (Q179X), in exon 8, leading to a stop codon and a heterozygous g.555C>A (C40X) recurrent nonsense mutation in exon 5. No mutations were found in patients with Peters' anomaly. We identified three mutations associated with aniridia phenotypes (Q179X, C40X, and V48fsX53). The three other mutations reported here cause non-aniridia ocular phenotypes associated in some cases with neurological anomalies. The IVS2+9G>A nucleotide change was detected in a patient with a microphthalmia phenotype. The L46P mutation was detected in a family with microphthalmia, cataract, and nystagmus. This mutation is located in the DNA-binding paired-domain and the crystallographic representations of this mutation show that this mutation may affect the helix-turn-helix motif, and as a consequence the DNA-binding properties of the resulting mutated protein. Ser74 is located in the PAX6 PD linker region, essential for DNA recognition and DNA binding, and the side chain of the Ser74 contributes to DNA recognition by the linker domain through direct contacts. Crystallographic representations show that the S74G mutation results in no side chain and therefore perturbs the DNA-binding properties of PAX6. This study highlights the severity and diversity of the consequences of PAX6 mutations that appeared to result from the complexity of the PAX6 gene structure, and the numerous possibilities for DNA binding. This study emphasizes the fact that neurodevelopmental abnormalities may be caused by PAX6 mutations. The neuro-developmental abnormalities caused by PAX6 mutations are probably still overlooked in the current clinical examinations performed throughout the world in patients affected by PAX6 mutations.
-
Dansault, Anouk; David, Gabriel; Schwartz, Claire; Jaliffa, Carolina; Vieira, Véronique; de la Houssaye, Guillaume; Bigot, Karine; Catin, Françise; Tattu, Laurent; Chopin, Catherine; Halimi, Philippe; Roche, Olivier; Van Regemorter, Nicole; Munier, Francis; Schorderet, Daniel; Dufier, Jean-Louis; Marsac, Cécile; Ricquier, Daniel; Menasche, Maurice; Penfornis, Alfred
2007-01-01
Purpose The PAX6 gene was first described as a candidate for human aniridia. However, PAX6 expression is not restricted to the eye and it appears to be crucial for brain development. We studied PAX6 mutations in a large spectrum of patients who presented with aniridia phenotypes, Peters' anomaly, and anterior segment malformations associated or not with neurological anomalies. Methods Patients and related families were ophthalmologically phenotyped, and in some cases neurologically and endocrinologically examined. We screened the PAX6 gene by direct sequencing in three groups of patients: those affected by aniridia; those with diverse ocular manifestations; and those with Peters' anomaly. Two mutations were investigated by generating crystallographic representations of the amino acid changes. Results Three novel heterozygous mutations affecting three unrelated families were identified: the g.572T>C nucleotide change, located in exon 5, and corresponding to the Leucine 46 Proline amino-acid mutation (L46P); the g.655A>G nucleotide change, located in exon 6, and corresponding to the Serine 74 Glycine amino-acid mutation (S74G); and the nucleotide deletion 579delG del, located in exon 6, which induces a frameshift mutation leading to a stop codon (V48fsX53). The L46P mutation was identified in affected patients presenting bilateral microphthalmia, cataracts, and nystagmus. The S74G mutation was found in a large family that had congenital ocular abnormalities, diverse neurological manifestations, and variable cognitive impairments. The 579delG deletion (V48fsX53) caused in the affected members of the same family bilateral aniridia associated with congenital cataract, foveal hypolasia, and nystagmus. We also detected a novel intronic nucleotide change, IVS2+9G>A (very likely a mutation) in an apparently isolated patient affected by a complex ocular phenotype, characterized primarily by a bilateral microphthalmia. Whether this nucleotide change is indeed pathogenic remains to be demonstrated. Two previously known heterozygous mutations of the PAX6 gene sequence were also detected in patients affected by aniridia: a de novo previously known nucleotide change, g.972C>T (Q179X), in exon 8, leading to a stop codon and a heterozygous g.555C>A (C40X) recurrent nonsense mutation in exon 5. No mutations were found in patients with Peters' anomaly. Conclusions We identified three mutations associated with aniridia phenotypes (Q179X, C40X, and V48fsX53). The three other mutations reported here cause non-aniridia ocular phenotypes associated in some cases with neurological anomalies. The IVS2+9G>A nucleotide change was detected in a patient with a microphthalmia phenotype. The L46P mutation was detected in a family with microphthalmia, cataract, and nystagmus. This mutation is located in the DNA-binding paired-domain and the crystallographic representations of this mutation show that this mutation may affect the helix-turn-helix motif, and as a consequence the DNA-binding properties of the resulting mutated protein. Ser74 is located in the PAX6 PD linker region, essential for DNA recognition and DNA binding, and the side chain of the Ser74 contributes to DNA recognition by the linker domain through direct contacts. Crystallographic representations show that the S74G mutation results in no side chain and therefore perturbs the DNA-binding properties of PAX6. This study highlights the severity and diversity of the consequences of PAX6 mutations that appeared to result from the complexity of the PAX6 gene structure, and the numerous possibilities for DNA binding. This study emphasizes the fact that neurodevelopmental abnormalities may be caused by PAX6 mutations. The neuro-developmental abnormalities caused by PAX6 mutations are probably still overlooked in the current clinical examinations performed throughout the world in patients affected by PAX6 mutations. PMID:17417613
-
Lahm, H; Hoeflich, A; Andre, S; Sordat, B; Kaltner, H; Wolf, E; Gabius, H J
2000-09-01
The family of Ca2+-independent galactoside-binding lectins with the beta-strand topology of the jelly-roll, referred to as galectins, is known to mediate and modulate a variety of cellular activities. Their functional versatility explains the current interest in monitoring their expression in cancer research, so far primarily focused on galectin-1 and -3. Tandem-repeat-type galectin-9 and its (most probably) allelic variant ecalectin, a potent eosinophil chemoattractant, are known to be human leukocyte products. We show by RT-PCR with primers specific for both that their mRNA is expressed in 17 of 21 human colorectal cancer lines. As also indicated by restriction analysis, in addition to the expected transcript of 571 bp an otherwise identical isoform coding for a 32-amino acid extension of the link peptide was detected. Positive cell lines differentially expressed either one (7 lines) or both transcripts (10 lines). Sequence analysis of RT-PCR products, performed in four cases, allowed to assign the standard transcript to ecalectin in the case of SW480 cells and detected two point mutations in the insert of the link peptide-coding sequence in WiDr and Colo205. Furthermore, this analysis identified the insertion of a single nucleotide into the coding sequence generating a frame-shift mutation, an event which has so far not been reported for any galectin. This alteration encountered in both transcripts of the WiDr line and the isoform transcript of Colo205 cells will most likely truncate the protein part within the second (C-terminal) carbohydrate recognition domain. Our results thus reveal the presence of mRNA for a galectin-9-isoform or a potent eosinophil chemoattractant (ecalectin) or a truncated version thereof with preserved N-terminal carbohydrate recognition domain in established human colon cancer cell lines.
-
DOE Office of Scientific and Technical Information (OSTI.GOV)
Ploos van Amstel, H.; Reitsma, P.H.; van der Logt, C.P.
The human protein S locus on chromosome 3 consists of two protein S genes, PS{alpha} and PS{beta}. Here the authors report the cloning and characterization of both genes. Fifteen exons of the PS{alpha} gene were identified that together code for protein S mRNA as derived from the reported protein S cDNAs. Analysis by primer extension of liver protein S mRNA, however, reveals the presence of two mRNA forms that differ in the length of their 5{prime}-noncoding region. Both transcripts contain a 5{prime}-noncoding region longer than found in the protein S cDNAs. The two products may arise from alternative splicing ofmore » an additional intron in this region or from the usage of two start sites for transcription. The intron-exon organization of the PS{alpha} gene fully supports the hypothesis that the protein S gene is the product of an evolutional assembling process in which gene modules coding for structural/functional protein units also found in other coagulation proteins have been put upstream of the ancestral gene of a steroid hormone binding protein. The PS{beta} gene is identified as a pseudogene. It contains a large variety of detrimental aberrations, viz., the absence of exon I, a splice site mutation, three stop codons, and a frame shift mutation. Overall the two genes PS{alpha} and PS{beta} show between their exonic sequences 96.5% homology. Southern analysis of primate DNA showed that the duplication of the ancestral protein S gene has occurred after the branching of the orangutan from the African apes. A nonsense mutation that is present in the pseudogene of man also could be identified in one of the two protein S genes of both chimpanzee and gorilla. This implicates that silencing of one of the two protein S genes must have taken place before the divergence of the three African apes.« less
-
El-Assaad, Atlal; Dawy, Zaher; Nemer, Georges
2015-01-01
Protein-DNA interaction is of fundamental importance in molecular biology, playing roles in functions as diverse as DNA transcription, DNA structure formation, and DNA repair. Protein-DNA association is also important in medicine; understanding Protein-DNA binding kinetics can assist in identifying disease root causes which can contribute to drug development. In this perspective, this work focuses on the transcription process by the GATA Transcription Factor (TF). GATA TF binds to DNA promoter region represented by `G,A,T,A' nucleotides sequence, and initiates transcription of target genes. When proper regulation fails due to some mutations on the GATA TF protein sequence or on the DNA promoter sequence (weak promoter), deregulation of the target genes might lead to various disorders. In this study, we aim to understand the electrostatic mechanism behind GATA TF and DNA promoter interactions, in order to predict Protein-DNA binding in the presence of mutations, while elaborating on non-covalent binding kinetics. To generate a family of mutants for the GATA:DNA complex, we replaced every charged amino acid, one at a time, with a neutral amino acid like Alanine (Ala). We then applied Poisson-Boltzmann electrostatic calculations feeding into free energy calculations, for each mutation. These calculations delineate the contribution to binding from each Ala-replaced amino acid in the GATA:DNA interaction. After analyzing the obtained data in view of a two-step model, we are able to identify potential key amino acids in binding. Finally, we applied the model to GATA-3:DNA (crystal structure with PDB-ID: 3DFV) binding complex and validated it against experimental results from the literature.
-
Valdramidou, Dimitra; Humphries, Martin J; Mould, A Paul
2008-11-21
Integrin-ligand interactions are regulated in a complex manner by divalent cations, and previous studies have identified ligand-competent, stimulatory, and inhibitory cation-binding sites. In collagen-binding integrins, such as alpha2beta1, ligand recognition takes place exclusively at the alpha subunit I domain. However, activation of the alphaI domain depends on its interaction with a structurally similar domain in the beta subunit known as the I-like or betaI domain. The top face of the betaI domain contains three cation-binding sites: the metal-ion dependent adhesion site (MIDAS), the ADMIDAS (adjacent to MIDAS), and LIMBS (ligand-associated metal-binding site). The role of these sites in controlling ligand binding to the alphaI domain has yet to be elucidated. Mutation of the MIDAS or LIMBS completely blocked collagen binding to alpha2beta1; in contrast mutation of the ADMIDAS reduced ligand recognition but this effect could be overcome by the activating monoclonal antibody TS2/16. Hence, the MIDAS and LIMBS appear to be essential for the interaction between alphaI and betaI, whereas occupancy of the ADMIDAS has an allosteric effect on the conformation of betaI. An activating mutation in the alpha2 I domain partially restored ligand binding to the MIDAS and LIMBS mutants. Analysis of the effects of Ca(2+), Mg(2+), and Mn(2+) on ligand binding to these mutants showed that the MIDAS is a ligand-competent site through which Mn(2+) stimulates ligand binding, whereas the LIMBS is a stimulatory Ca(2+)-binding site, occupancy of which increases the affinity of Mg(2+) for the MIDAS.
-
Liu, Yingjie; Larsen, Kamilla Taunsig; Nani, Alma; Tian, Xixi; Holt, Christian; Wang, Ruiwu; Fill, Michael
2017-01-01
A number of point mutations in the intracellular Ca2+-sensing protein calmodulin (CaM) are arrhythmogenic, yet their underlying mechanisms are not clear. These mutations generally decrease Ca2+ binding to CaM and impair inhibition of CaM-regulated Ca2+ channels like the cardiac Ca2+ release channel (ryanodine receptor, RyR2), and it appears that attenuated CaM Ca2+ binding correlates with impaired CaM-dependent RyR2 inhibition. Here, we investigated the RyR2 inhibitory action of the CaM p.Phe142Leu mutation (F142L; numbered including the start-Met), which markedly reduces CaM Ca2+ binding. Surprisingly, CaM-F142L had little to no aberrant effect on RyR2-mediated store overload-induced Ca2+ release in HEK293 cells compared with CaM-WT. Furthermore, CaM-F142L enhanced CaM-dependent RyR2 inhibition at the single channel level compared with CaM-WT. This is in stark contrast to the actions of arrhythmogenic CaM mutations N54I, D96V, N98S, and D130G, which all diminish CaM-dependent RyR2 inhibition. Thermodynamic analysis showed that apoCaM-F142L converts an endothermal interaction between CaM and the CaM-binding domain (CaMBD) of RyR2 into an exothermal one. Moreover, NMR spectra revealed that the CaM-F142L-CaMBD interaction is structurally different from that of CaM-WT at low Ca2+. These data indicate a distinct interaction between CaM-F142L and the RyR2 CaMBD, which may explain the stronger CaM-dependent RyR2 inhibition by CaM-F142L, despite its reduced Ca2+ binding. Collectively, these results add to our understanding of CaM-dependent regulation of RyR2 as well as the mechanistic effects of arrhythmogenic CaM mutations. The unique properties of the CaM-F142L mutation may provide novel clues on how to suppress excessive RyR2 Ca2+ release by manipulating the CaM-RyR2 interaction. PMID:27927985
-
Dissection of specific binding of HIV-1 Gag to the 'packaging signal' in viral RNA.
Comas-Garcia, Mauricio; Datta, Siddhartha Ak; Baker, Laura; Varma, Rajat; Gudla, Prabhakar R; Rein, Alan
2017-07-20
Selective packaging of HIV-1 genomic RNA (gRNA) requires the presence of a cis -acting RNA element called the 'packaging signal' (Ψ). However, the mechanism by which Ψ promotes selective packaging of the gRNA is not well understood. We used fluorescence correlation spectroscopy and quenching data to monitor the binding of recombinant HIV-1 Gag protein to Cy5-tagged 190-base RNAs. At physiological ionic strength, Gag binds with very similar, nanomolar affinities to both Ψ-containing and control RNAs. We challenged these interactions by adding excess competing tRNA; introducing mutations in Gag; or raising the ionic strength. These modifications all revealed high specificity for Ψ. This specificity is evidently obscured in physiological salt by non-specific, predominantly electrostatic interactions. This nonspecific activity was attenuated by mutations in the MA, CA, and NC domains, including CA mutations disrupting Gag-Gag interaction. We propose that gRNA is selectively packaged because binding to Ψ nucleates virion assembly with particular efficiency.
-
Parkin binds the Rpn10 subunit of 26S proteasomes through its ubiquitin-like domain
Sakata, Eri; Yamaguchi, Yoshiki; Kurimoto, Eiji; Kikuchi, Jun; Yokoyama, Shigeyuki; Yamada, Shingo; Kawahara, Hiroyuki; Yokosawa, Hideyoshi; Hattori, Nobutaka; Mizuno, Yoshikuni; Tanaka, Keiji; Kato, Koichi
2003-01-01
Parkin, a product of the causative gene of autosomal-recessive juvenile parkinsonism (AR-JP), is a RING-type E3 ubiquitin ligase and has an amino-terminal ubiquitin-like (Ubl) domain. Although a single mutation that causes an Arg to Pro substitution at position 42 of the Ubl domain (the Arg 42 mutation) has been identified in AR-JP patients, the function of this domain is not clear. In this study, we determined the three-dimensional structure of the Ubl domain of parkin by NMR, in particular by extensive use of backbone 15N-1H residual dipolar-coupling data. Inspection of chemical-shift-perturbation data showed that the parkin Ubl domain binds the Rpn10 subunit of 26S proteasomes via the region of parkin that includes position 42. Our findings suggest that the Arg 42 mutation induces a conformational change in the Rpn10-binding site of Ubl, resulting in impaired proteasomal binding of parkin, which could be the cause of AR-JP. PMID:12634850
-
Liu, Fengjiao; Zhang, John Z H; Mei, Ye
2016-06-01
Previous experimental study measuring the binding affinities of biotin to the wild type streptavidin (WT) and three mutants (S45A, D128A and S45A/D128A double mutant) has shown that the loss of binding affinity from the double mutation is larger than the direct sum of those from two single mutations. The origin of this cooperativity has been investigated in this work through molecular dynamics simulations and the end-state free energy method using the polarized protein-specific charge. The results show that this cooperativity comes from both the enthalpy and entropy contributions. The former contribution mainly comes from the alternations of solvation free energy. Decomposition analysis shows that the mutated residues nearly have no contributions to the cooperativity. Instead, N49 and S88, which are located at the entry of the binding pocket and interact with the carboxyl group of biotin, make the dominant contribution among all the residues in the first binding shell around biotin.
-
NASA Astrophysics Data System (ADS)
Liu, Fengjiao; Zhang, John Z. H.; Mei, Ye
2016-06-01
Previous experimental study measuring the binding affinities of biotin to the wild type streptavidin (WT) and three mutants (S45A, D128A and S45A/D128A double mutant) has shown that the loss of binding affinity from the double mutation is larger than the direct sum of those from two single mutations. The origin of this cooperativity has been investigated in this work through molecular dynamics simulations and the end-state free energy method using the polarized protein-specific charge. The results show that this cooperativity comes from both the enthalpy and entropy contributions. The former contribution mainly comes from the alternations of solvation free energy. Decomposition analysis shows that the mutated residues nearly have no contributions to the cooperativity. Instead, N49 and S88, which are located at the entry of the binding pocket and interact with the carboxyl group of biotin, make the dominant contribution among all the residues in the first binding shell around biotin.
-
Chan, Kwok Keung; Wong, Corinne Kung Yen; Lui, Vincent Chi Hang; Tam, Paul Kwong Hang; Sham, Mai Har
2003-10-15
SOX10 is a member of the SOX gene family related by homology to the high-mobility group (HMG) box region of the testis-determining gene SRY. Mutations of the transcription factor gene SOX10 lead to Waardenburg-Hirschsprung syndrome (Waardenburg-Shah syndrome, WS4) in humans. A number of SOX10 mutations have been identified in WS4 patients who suffer from different extents of intestinal aganglionosis, pigmentation, and hearing abnormalities. Some patients also exhibit signs of myelination deficiency in the central and peripheral nervous systems. Although the molecular bases for the wide range of symptoms displayed by the patients are still not clearly understood, a few target genes for SOX10 have been identified. We have analyzed the impact of six different SOX10 mutations on the activation of SOX10 target genes by yeast one-hybrid and mammalian cell transfection assays. To investigate the transactivation activities of the mutant proteins, three different SOX target binding sites were introduced into luciferase reporter gene constructs and examined in our series of transfection assays: consensus HMG domain protein binding sites; SOX10 binding sites identified in the RET promoter; and Sox10 binding sites identified in the P0 promoter. We found that the same mutation could have different transactivation activities when tested with different target binding sites and in different cell lines. The differential transactivation activities of the SOX10 mutants appeared to correlate with the intestinal and/or neurological symptoms presented in the patients. Among the six mutant SOX10 proteins tested, much reduced transactivation activities were observed when tested on the SOX10 binding sites from the RET promoter. Of the two similar mutations X467K and 1400del12, only the 1400del12 mutant protein exhibited an increase of transactivation through the P0 promoter. While the lack of normal SOX10 mediated activation of RET transcription may lead to intestinal aganglionosis, overexpression of genes coding for structural myelin proteins such as P0 due to mutant SOX10 may explain the dysmyelination phenotype observed in the patients with an additional neurological disorder. Copyright 2003 Wiley-Liss, Inc.
-
Redesign of LAOBP to bind novel l-amino acid ligands.
Banda-Vázquez, Jesús; Shanmugaratnam, Sooruban; Rodríguez-Sotres, Rogelio; Torres-Larios, Alfredo; Höcker, Birte; Sosa-Peinado, Alejandro
2018-05-01
Computational protein design is still a challenge for advancing structure-function relationships. While recent advances in this field are promising, more information for genuine predictions is needed. Here, we discuss different approaches applied to install novel glutamine (Gln) binding into the Lysine/Arginine/Ornithine binding protein (LAOBP) from Salmonella typhimurium. We studied the ligand binding behavior of two mutants: a binding pocket grafting design based on a structural superposition of LAOBP to the Gln binding protein QBP from Escherichia coli and a design based on statistical coupled positions. The latter showed the ability to bind Gln even though the protein was not very stable. Comparison of both approaches highlighted a nonconservative shared point mutation between LAOBP_graft and LAOBP_sca. This context dependent L117K mutation in LAOBP turned out to be sufficient for introducing Gln binding, as confirmed by different experimental techniques. Moreover, the crystal structure of LAOBP_L117K in complex with its ligand is reported. © 2018 The Protein Society.
-
Ido, Kunio; Kakiuchi, Shusuke; Uno, Chihiro; Nishimura, Taishi; Fukao, Yoichiro; Noguchi, Takumi; Sato, Fumihiko; Ifuku, Kentaro
2012-07-27
The PsbP protein regulates the binding properties of Ca(2+) and Cl(-), and stabilizes the Mn cluster of photosystem II (PSII); however, the binding site and topology in PSII have yet to be clarified. Here we report that the structure around His-144 and Asp-165 in PsbP, which is suggested to be a metal binding site, has a crucial role for the functional interaction between PsbP and PSII. The mutated PsbP-H144A protein exhibits reduced ability to retain Cl(-) anions in PSII, whereas the D165V mutation does not affect PsbP function. Interestingly, H144A/D165V double mutation suppresses the effect of H144A mutation, suggesting that these residues have a role other than metal binding. FTIR difference spectroscopy suggests that H144A/D165V restores proper interaction with PSII and induces the conformational change around the Mn cluster during the S(1)/S(2) transition. Cross-linking experiments show that the H144A mutation affects the direct interaction between PsbP and the Cyt b(559) α subunit of PSII (the PsbE protein). However, this interaction is restored in the H144A/D165V mutant. In the PsbP structure, His-144 and Asp-165 form a salt bridge. H144A mutation is likely to disrupt this bridge and liberate Asp-165, inhibiting the proper PsbP-PSII interaction. Finally, mass spectrometric analysis has identified the cross-linked sites of PsbP and PsbE as Ala-1 and Glu-57, respectively. Therefore His-144, in the C-terminal domain of PsbP, plays a crucial role in maintaining proper N terminus interaction. These data provide important information about the binding characteristics of PsbP in green plant PSII.
-
Rannversson, Hafsteinn; Wilson, Pamela; Kristensen, Kristina Birch; Sinning, Steffen; Kristensen, Anders Skov; Strømgaard, Kristian; Andersen, Jacob
2015-01-01
The serotonin transporter (SERT) terminates serotonergic neurotransmission by performing reuptake of released serotonin, and SERT is the primary target for antidepressants. SERT mediates the reuptake of serotonin through an alternating access mechanism, implying that a central substrate site is connected to both sides of the membrane by permeation pathways, of which only one is accessible at a time. The coordinated conformational changes in SERT associated with substrate translocation are not fully understood. Here, we have identified a Leu to Glu mutation at position 406 (L406E) in the extracellular loop 4 (EL4) of human SERT, which induced a remarkable gain-of-potency (up to >40-fold) for a range of SERT inhibitors. The effects were highly specific for L406E relative to six other mutations in the same position, including the closely related L406D mutation, showing that the effects induced by L406E are not simply charge-related effects. Leu406 is located >10 Å from the central inhibitor binding site indicating that the mutation affects inhibitor binding in an indirect manner. We found that L406E decreased accessibility to a residue in the cytoplasmic pathway. The shift in equilibrium to favor a more outward-facing conformation of SERT can explain the reduced turnover rate and increased association rate of inhibitor binding we found for L406E. Together, our findings show that EL4 allosterically can modulate inhibitor binding within the central binding site, and substantiates that EL4 has an important role in controlling the conformational equilibrium of human SERT. PMID:25903124
-
RUCS: rapid identification of PCR primers for unique core sequences.
Thomsen, Martin Christen Frølund; Hasman, Henrik; Westh, Henrik; Kaya, Hülya; Lund, Ole
2017-12-15
Designing PCR primers to target a specific selection of whole genome sequenced strains can be a long, arduous and sometimes impractical task. Such tasks would benefit greatly from an automated tool to both identify unique targets, and to validate the vast number of potential primer pairs for the targets in silico. Here we present RUCS, a program that will find PCR primer pairs and probes for the unique core sequences of a positive genome dataset complement to a negative genome dataset. The resulting primer pairs and probes are in addition to simple selection also validated through a complex in silico PCR simulation. We compared our method, which identifies the unique core sequences, against an existing tool called ssGeneFinder, and found that our method was 6.5-20 times more sensitive. We used RUCS to design primer pairs that would target a set of genomes known to contain the mcr-1 colistin resistance gene. Three of the predicted pairs were chosen for experimental validation using PCR and gel electrophoresis. All three pairs successfully produced an amplicon with the target length for the samples containing mcr-1 and no amplification products were produced for the negative samples. The novel methods presented in this manuscript can reduce the time needed to identify target sequences, and provide a quick virtual PCR validation to eliminate time wasted on ambiguously binding primers. Source code is freely available on https://bitbucket.org/genomicepidemiology/rucs. Web service is freely available on https://cge.cbs.dtu.dk/services/RUCS. mcft@cbs.dtu.dk. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.
-
Kim, Jiae; Roberts, Anne; Yuan, Hua; Xiong, Yong; Anderson, Karen S.
2012-01-01
Human immunodeficiency virus type-1 (HIV-1) requires reverse transcriptase (RT) and HIV-1 nucleocapsid protein (NCp7) for proper viral replication. HIV-1 NCp7 has been shown to enhance various steps in reverse transcription including tRNA initiation and strand transfer which may be mediated through interactions with RT as well as RNA and DNA oligonucleotides. With the use of DNA oligonucleotides, we have examined the interaction of NCp7 with RT and the kinetics of reverse transcription during (+)-strand synthesis with an NCp7-facilitated annealed primer-template. Using a pre-steady state kinetics approach, the NCp7-annealed primer-template has a substantial increase (3-7 fold) in the rate of incorporation (kpol) by RT as compared to heat annealed primer-template with single nucleotide incorporation. There was also a 2-fold increase in the binding affinity constant (Kd) of the nucleotide. These differences in kpol and Kd were not through direct interactions between HIV-1 RT and NCp7. When examining extension by RT, the data suggests that the NCp7-annealed primer-template facilitates the formation of a longer product more quickly compared to the heat annealed primer-template. This enhancement in rate is mediated through interactions with NCp7’s zinc fingers and N-terminal domain and nucleic acids. The NCp7-annealed primer-template also enhances the fidelity of RT (3-fold) by slowing the rate of incorporation of an incorrect nucleotide. Taken together, this study elucidates a new role of NCp7 by facilitating DNA-directed DNA synthesis during reverse transcription by HIV-1 RT that may translate into enhanced viral fitness and offers an avenue to exploit for targeted therapeutic intervention against HIV. PMID:22210155
-
Amylin S20G mutation in Mexican population.
Garcia-Gonzalez, Claudia Lorena; Montoya-Fuentes, Hector; Padilla-Rosas, Miguel; Sanchez-Corona, Jose
2007-04-01
Diabetes Mellitus type 2 (DM2) is a group of metabolic disorders characterized by defective insulin action or secretion or both with a 10.6% incidence in Mexican Mestizo population, DM2 is also classified within the localized misfolding diseases due to the amyloid pancreatic deposits found in 90% of the DM2 necropsies. The pancreatic amyloid main component is a protein known as human islet amyloid polypeptide (hIAPP) or amylin, the most common mutation is the S20G in Asian population with a polymorphic frequency in DM2 Asian patients. The aim of this study was to search this mutation in Mexican Mestizo general population (104) and DM2 patients (100). This is the first molecular study of hIAPP gene in Mexican population and in which we developed an alternative more effective antisense primer for the analysis of the NFGAILSS region in hIAPP exon 3 critical for the amyloid beta structure formation. We did not find the mutation in any of the 204 analyzed samples, thus the findings show that S20G is not a common mutation in Mexican Mestizo population.
-
NASA Astrophysics Data System (ADS)
Su, Qiang; Zhou, Xiaoming
2008-12-01
Many pathogenic and genetic diseases are associated with changes in the sequence of particular genes. We describe here a rapid and highly efficient assay for the detection of point mutation. This method is a combination of isothermal rolling circle amplification (RCA) and high sensitive electrochemluminescence (ECL) detection. In the design, a circular template generated by ligation upon the recognition of a point mutation on DNA targets was amplified isothermally by the Phi29 polymerase using a biotinylated primer. The elongation products were hybridized with tris (bipyridine) ruthenium (TBR)-tagged probes and detected in a magnetic bead based ECL platform, indicating the mutation occurrence. P53 was chosen as a model for the identification of this method. The method allowed sensitive determination of the P53 mutation from wild-type and mutant samples. The main advantage of RCA-ECL is that it can be performed under isothermal conditions and avoids the generation of false-positive results. Furthermore, ECL provides a faster, more sensitive, and economical option to currently available electrophoresis-based methods.
-
Suzuki, Tamio; Miyamura, Yoshinori; Inagaki, Katsuhiko; Tomita, Yasushi
2003-08-01
Oculocutaneous albinisms (OCAs) are due to various gene mutations that cause a disruption of melanogenesis in the melanocyte. Four different genes associated with human OCA have been reported, however, not all of OCA patients can be classified according to these four genes. We have sought to find a new major locus for Japanese OCA. Recently two genes, RAB38 and RAB7, were reported to play an important role in melanogenesis in the melanocyte, suggesting that these two genes could be good candidates for new OCA loci. To determine the structures of the human RAB38 and RAB7 genes, and examine if the two genes are new major loci for Japanese OCA. We screened mutations in these genes of 25 Japanese OCA patients who lacked mutations in the OCA1 and OCA2 genes with SSCP/heteroduplexes method. We determined the both genes, and their genomic organizations to design the primers for SSCP/heteroduplexes method. And then we screened mutations, but no mutation was detected. Neither of the genes is a new major locus for Japanese OCA.
-
Weber, J Mark; Reeves, Andrew; Cernota, William H; Wesley, Roy K
2017-01-01
Transposon mutagenesis is an invaluable technique in molecular biology for the creation of random mutations that can be easily identified and mapped. However, in the field of microbial strain improvement, transposon mutagenesis has scarcely been used; instead, chemical and physical mutagenic methods have been traditionally favored. Transposons have the advantage of creating single mutations in the genome, making phenotype to genotype assignments less challenging than with traditional mutagens which commonly create multiple mutations in the genome. The site of a transposon mutation can also be readily mapped using DNA sequencing primer sites engineered into the transposon termini. In this chapter an in vitro method for transposon mutagenesis of Saccharopolyspora erythraea is presented. Since in vivo transposon tools are not available for most actinomycetes including S. erythraea, an in vitro method was developed. The in vitro method involves a significant investment in time and effort to create the mutants, but once the mutants are made and screened, a large number of highly relevant mutations of direct interest to erythromycin production can be found.
-
Boyoglu-Barnum, S; Todd, S O; Meng, J; Barnum, T R; Chirkova, T; Haynes, L M; Jadhao, S J; Tripp, R A; Oomens, A G; Moore, M L; Anderson, L J
2017-05-15
Respiratory syncytial virus (RSV) belongs to the family Paramyxoviridae and is the single most important cause of serious lower respiratory tract infections in young children, yet no highly effective treatment or vaccine is available. Through a CX3C chemokine motif ( 182 CWAIC 186 ) in the G protein, RSV binds to the corresponding chemokine receptor, CX3CR1. Since RSV binding to CX3CR1 contributes to disease pathogenesis, we investigated whether a mutation in the CX3C motif by insertion of an alanine, A 186 , within the CX3C motif, mutating it to CX4C ( 182 CWAIAC 187 ), which is known to block binding to CX3CR1, might decrease disease. We studied the effect of the CX4C mutation in two strains of RSV (A2 and r19F) in a mouse challenge model. We included RSV r19F because it induces mucus production and airway resistance, two manifestations of RSV infection in humans, in mice. Compared to wild-type (wt) virus, mice infected with CX4C had a 0.7 to 1.2 log 10 -fold lower virus titer in the lung at 5 days postinfection (p.i.) and had markedly reduced weight loss, pulmonary inflammatory cell infiltration, mucus production, and airway resistance after challenge. This decrease in disease was not dependent on decrease in virus replication but did correspond to a decrease in pulmonary Th2 and inflammatory cytokines. Mice infected with CX4C viruses also had higher antibody titers and a Th1-biased T cell memory response at 75 days p.i. These results suggest that the CX4C mutation in the G protein could improve the safety and efficacy of a live attenuated RSV vaccine. IMPORTANCE RSV binds to the corresponding chemokine receptor, CX3CR1, through a CX3C chemokine motif ( 182 CWAIC 186 ) in the G protein. RSV binding to CX3CR1 contributes to disease pathogenesis; therefore, we investigated whether a mutation in the CX3C motif by insertion of an alanine, A 186 , within the CX3C motif, mutating it to CX4C ( 182 CWAIAC 187 ), known to block binding to CX3CR1, might decrease disease. The effect of this mutation and treatment with the F(ab') 2 form of the anti-RSV G 131-2G monoclonal antibody (MAb) show that mutating the CX3C motif to CX4C blocks much of the disease and immune modulation associated with the G protein and should improve the safety and efficacy of a live attenuated RSV vaccine. Copyright © 2017 American Society for Microbiology.
-
Mutation analysis and molecular modeling for the investigation of ligand-binding modes of GPR84.
Nikaido, Yoshiaki; Koyama, Yuuta; Yoshikawa, Yasushi; Furuya, Toshio; Takeda, Shigeki
2015-05-01
GPR84 is a G protein-coupled receptor for medium-chain fatty acids. Capric acid and 3,3'-diindolylmethane are specific agonists for GPR84. We built a homology model of a GPR84-capric acid complex to investigate the ligand-binding mode using the crystal structure of human active-state β2-adrenergic receptor. We performed site-directed mutagenesis to subject ligand-binding sites to our model using GPR84-Giα fusion proteins and a [(35)S]GTPγS-binding assay. We compared the activity of the wild type and mutated forms of GPR84 by [(35)S]GTPγS binding to capric acid and diindolylmethane. The mutations L100D `Ballesteros-Weinstein numbering: 3.32), F101Y (3.33) and N104Q (3.36) in the transmembrane helix III and N357D (7.39) in the transmembrane helix VII resulted in reduced capric acid activity but maintained the diindolylmethane responses. Y186F (5.46) and Y186H (5.46) mutations had no characteristic effect on capric acid but with diindolylmethane they significantly affected the G protein activation efficiency. The L100D (3.32) mutant responded to decylamine, a fatty amine, instead of a natural agonist, the fatty acid capric acid, suggesting that we have identified a mutated G protein-coupled receptor-artificial ligand pairing. Our molecular model provides an explanation for these results and interactions between GPR84 and capric acid. Further, from the results of a double stimulation assay, we concluded that diindolylmethane was a positive allosteric modulator for GPR84. © The Authors 2014. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.
-
German-Retana, Sylvie; Walter, Jocelyne; Doublet, Bénédicte; Roudet-Tavert, Geneviève; Nicaise, Valérie; Lecampion, Cécile; Houvenaghel, Marie-Christine; Robaglia, Christophe; Michon, Thierry; Le Gall, Olivier
2008-01-01
The eukaryotic translation initiation factor 4E (eIF4E) (the cap-binding protein) is involved in natural resistance against several potyviruses in plants. In lettuce, the recessive resistance genes mo11 and mo12 against Lettuce mosaic virus (LMV) are alleles coding for forms of eIF4E unable, or less effective, to support virus accumulation. A recombinant LMV expressing the eIF4E of a susceptible lettuce variety from its genome was able to produce symptoms in mo11 or mo12 varieties. In order to identify the eIF4E amino acid residues necessary for viral infection, we constructed recombinant LMV expressing eIF4E with point mutations affecting various amino acids and compared the abilities of these eIF4E mutants to complement LMV infection in resistant plants. Three types of mutations were produced in order to affect different biochemical functions of eIF4E: cap binding, eIF4G binding, and putative interaction with other virus or host proteins. Several mutations severely reduced the ability of eIF4E to complement LMV accumulation in a resistant host and impeded essential eIF4E functions in yeast. However, the ability of eIF4E to bind a cap analogue or to fully interact with eIF4G appeared unlinked to LMV infection. In addition to providing a functional mutational map of a plant eIF4E, this suggests that the role of eIF4E in the LMV cycle might be distinct from its physiological function in cellular mRNA translation. PMID:18480444
-
Lin, Xiaoyan; Ruiz, Janelle; Bajraktari, Ilda; Ohman, Rachel; Banerjee, Soojay; Gribble, Katherine; Kaufman, Joshua D.; Wingfield, Paul T.; Griggs, Robert C.; Fischbeck, Kenneth H.; Mankodi, Ami
2014-01-01
The core of skeletal muscle Z-discs consists of actin filaments from adjacent sarcomeres that are cross-linked by α-actinin homodimers. Z-disc-associated, alternatively spliced, PDZ motif-containing protein (ZASP)/Cypher interacts with α-actinin, myotilin, and other Z-disc proteins via the PDZ domain. However, these interactions are not sufficient to maintain the Z-disc structure. We show that ZASP directly interacts with skeletal actin filaments. The actin-binding domain is between the modular PDZ and LIM domains. This ZASP region is alternatively spliced so that each isoform has unique actin-binding domains. All ZASP isoforms contain the exon 6-encoded ZASP-like motif that is mutated in zaspopathy, a myofibrillar myopathy (MFM), whereas the exon 8–11 junction-encoded peptide is exclusive to the postnatal long ZASP isoform (ZASP-LΔex10). MFM is characterized by disruption of skeletal muscle Z-discs and accumulation of myofibrillar degradation products. Wild-type and mutant ZASP interact with α-actin, α-actinin, and myotilin. Expression of mutant, but not wild-type, ZASP leads to Z-disc disruption and F-actin accumulation in mouse skeletal muscle, as in MFM. Mutations in the actin-binding domain of ZASP-LΔex10, but not other isoforms, cause disruption of the actin cytoskeleton in muscle cells. These isoform-specific mutation effects highlight the essential role of the ZASP-LΔex10 isoform in F-actin organization. Our results show that MFM-associated ZASP mutations in the actin-binding domain have deleterious effects on the core structure of the Z-discs in skeletal muscle. PMID:24668811
-
Tanneeru, Karunakar; Guruprasad, Lalitha
2013-11-01
Aurora-A, B and C are non-receptor serine/threonine kinases in Homo sapiens. In spite of high similarity in their sequences, they possess distinct binding partners. These kinases play an important role in cell division and overexpressed in certain cancers. It has been demonstrated that Gly198 in Aurora-A kinase is responsible for its basal kinase activity, the mutation G198N transforms Aurora-A to Aurora-B like function and localization by binding to Inner centromere protein (INCENP). The molecular mechanisms, structural determinants and the binding energetics of the Aurora-A - INCENP complex owing to a single amino acid G198N mutation are not studied. Therefore, we have docked INCENP into human Aurora-A kinase, mutated Gly198 to Asn, Leu and Ala. The wild type and mutant Aurora-A - INCENP complexes were subjected to 40 ns molecular dynamics (MD) simulations. The Asn198 is located in the amphipathic cavity comprising Leu869(IN), Glu868(IN), Thr872(IN), Tyr197(AurA) and Tyr199(AurA) and the interactions mediated via hydrogen bonds are important to stabilize the Aurora-A(G198N) - INCENP complex. The fluctuations in the secondary structural elements and the solvent accessible surface area of all the four complexes during the MD simulations were studied. We calculated the binding free energy upon mutation in the three mutant complexes. The Aurora-A(G198N) - INCENP complex with hydrophilic amino acid mutation has the negative free energy of solvation indicating favorable interactions with INCENP. Our results provide the structural basis and energetics of the human Aurora-A(G198N) - INCENP complex.
-
High Resolution Analysis of Copy Number Mutation in Breast Cancer
2005-05-01
tissues and Epstein - Barr sentations and arrays of Hind III probes additional CNPs, as would an increase in the virus -immortalized lymphoblastoid cell...software and laboratory procedures for the design of inter-phase FISH primers. We have also made progress in developing database and data processing...Cancer progression often involves alterations in DNA copy number. Newly developed microarray technologies enable simultane- ous measurement of copy
-
Schmoock, Gernot; Elschner, Mandy; Sprague, Lisa D
2015-03-07
A frame-shift mutation in the flagellum motor gene motB coding for the chemotaxis MotB protein of Burkholderia mallei has been utilized to design a conventional duplex PCR assay with fluorescent labelled primers. Species specificity was tested with a panel of 13 Burkholderia type strains. A total of 41 B. mallei field strains, 36 B. pseudomallei field strains, and 1 B. thailandensis field strain from different geographic regions were tested and correctly identified. Testing of 55 non-Burkholderia bacterial species revealed 100% specificity of the assay. The minimum detection limit was 1 pg DNA or 160 GE for B. mallei and 130 GE for B. pseudomallei, respectively. This assay enables the clear distinction between B. mallei and B. pseudomallei/B. thailandensis.
-
Hersh, Bradley M
2016-12-01
SummaryA single gene might have several functions within an organism, and so mutational loss of that gene has multiple effects across different physiological systems in the organism. Though the white gene in Drosophila melanogaster was identified originally for its effect on fly eye color, an article by Xiao and Robertson in the June 2016 issue of GENETICS describes a function for the white gene in the response of Drosophila to oxygen deprivation. This Primer article provides background information on the white gene, the phenomenon of pleiotropy, and the molecular and genetic approaches used in the study to demonstrate a new behavioral function for the white gene. Copyright © 2016 by the Genetics Society of America.
-
2004-01-01
Abstract The purpose of this study was to evaluate the cationic trypsinogen gene in miniature schnauzers for possible mutations. Genetic mutations have been linked with hereditary pancreatitis in humans. Four miniature schnauzers were selected on the basis of a clinical history of pancreatitis. One healthy miniature schnauzer and 1 healthy mixed breed canine were enrolled as controls. DNA was extracted from these canines using a commercial kit. Primers were designed to amplify the entire canine cationic trypsinogen cDNA sequence. A polymerase chain reaction (PCR) was performed and products were purified and sequenced. All sequences were then compared. The healthy control canine, a healthy miniature schnauzer, and the 4 miniature schnauzers with pancreatitis showed identical sequences of the cationic trypsinogen gene to the published sequence. We conclude that, in contrast to humans with hereditary pancreatitis, mutations of the cationic trypsinogen gene do not play a major role in the genesis of pancreatitis in the miniature schnauzer. PMID:15581228
-
Bishop, Micah A; Steiner, Jörg M; Moore, Lisa E; Williams, David A
2004-10-01
The purpose of this study was to evaluate the cationic trypsinogen gene in miniature schnauzers for possible mutations. Genetic mutations have been linked with hereditary pancreatitis in humans. Four miniature schnauzers were selected on the basis of a clinical history of pancreatitis. One healthy miniature schnauzer and 1 healthy mixed breed canine were enrolled as controls. DNA was extracted from these canines using a commercial kit. Primers were designed to amplify the entire canine cationic trypsinogen cDNA sequence. A polymerase chain reaction (PCR) was performed and products were purified and sequenced. All sequences were then compared. The healthy control canine, a healthy miniature schnauzer, and the 4 miniature schnauzers with pancreatitis showed identical sequences of the cationic trypsinogen gene to the published sequence. We conclude that, in contrast to humans with hereditary pancreatitis, mutations of the cationic trypsinogen gene do not play a major role in the genesis of pancreatitis in the miniature schnauzer.
-
Pick Your Poisson: An Educational Primer for Luria and Delbrück's Classic Paper.
Meneely, Philip M
2016-02-01
The origin of beneficial mutations is fundamentally important in understanding the processes by which natural selection works. Using phage-resistant mutants in Escherichia coli as their model for identifying the origin of beneficial mutations, Luria and Delbrück distinguished between two different hypotheses. Under the first hypothesis, which they termed "acquired immunity," the phages induced bacteria to mutate to immunity; this predicts that none of the resistant mutants were present before infection by the phages. Under the second hypothesis, termed "mutation to immunity," resistant bacteria arose from random mutations independent of the presence of the phages; this predicts that resistant bacteria were present in the population before infection by the phages. These two hypotheses could be distinguished by calculating the frequencies at which resistant mutants arose in separate cultures infected at the same time and comparing these frequencies to the theoretical results under each model. The data clearly show that mutations arise at a frequency that is independent of the presence of the phages. By inference, natural selection reveals the genetic variation that is present in a population rather than inducing or causing this variation. Copyright © 2016 by the Genetics Society of America.
-
Duan, Yabing; Zhang, Xiaoke; Ge, Changyan; Wang, Yong; Cao, Junhong; Jia, Xiaojing; Wang, Jianxin; Zhou, Mingguo
2014-01-01
Resistance of Fusarium graminearum to carbendazim is caused by point mutations in the β2-tubulin gene. The point mutation at codon 167 (TTT → TAT, F167Y) occurs in more than 90% of field resistant isolates in China. To establish a suitable method for rapid detection of the F167Y mutation in F. graminearum, an efficient and simple method with high specificity was developed based on loop-mediated isothermal amplification (LAMP). A set of four primers was designed and optimized to specially distinguish the F167Y mutation genotype. The LAMP reaction was optimal at 63°C for 60 min. When hydroxynaphthol blue dye (HNB) was added prior to amplification, samples with DNA of the F167Y mutation developed a characteristic sky blue color after the reaction but those without DNA or with different DNA did not. Results of HNB staining method were reconfirmed by gel electrophoresis. The developed LAMP had good specificity, stability and repeatability and was suitable for monitoring carbendazim-resistance populations of F. graminearum in agricultural production. PMID:25403277
-
Zhou, Shuntai; Jones, Corbin; Mieczkowski, Piotr
2015-01-01
ABSTRACT Validating the sampling depth and reducing sequencing errors are critical for studies of viral populations using next-generation sequencing (NGS). We previously described the use of Primer ID to tag each viral RNA template with a block of degenerate nucleotides in the cDNA primer. We now show that low-abundance Primer IDs (offspring Primer IDs) are generated due to PCR/sequencing errors. These artifactual Primer IDs can be removed using a cutoff model for the number of reads required to make a template consensus sequence. We have modeled the fraction of sequences lost due to Primer ID resampling. For a typical sequencing run, less than 10% of the raw reads are lost to offspring Primer ID filtering and resampling. The remaining raw reads are used to correct for PCR resampling and sequencing errors. We also demonstrate that Primer ID reveals bias intrinsic to PCR, especially at low template input or utilization. cDNA synthesis and PCR convert ca. 20% of RNA templates into recoverable sequences, and 30-fold sequence coverage recovers most of these template sequences. We have directly measured the residual error rate to be around 1 in 10,000 nucleotides. We use this error rate and the Poisson distribution to define the cutoff to identify preexisting drug resistance mutations at low abundance in an HIV-infected subject. Collectively, these studies show that >90% of the raw sequence reads can be used to validate template sampling depth and to dramatically reduce the error rate in assessing a genetically diverse viral population using NGS. IMPORTANCE Although next-generation sequencing (NGS) has revolutionized sequencing strategies, it suffers from serious limitations in defining sequence heterogeneity in a genetically diverse population, such as HIV-1 due to PCR resampling and PCR/sequencing errors. The Primer ID approach reveals the true sampling depth and greatly reduces errors. Knowing the sampling depth allows the construction of a model of how to maximize the recovery of sequences from input templates and to reduce resampling of the Primer ID so that appropriate multiplexing can be included in the experimental design. With the defined sampling depth and measured error rate, we are able to assign cutoffs for the accurate detection of minority variants in viral populations. This approach allows the power of NGS to be realized without having to guess about sampling depth or to ignore the problem of PCR resampling, while also being able to correct most of the errors in the data set. PMID:26041299
-
Hamrick, Terri S.; Harris, Sandra L.; Spears, Patricia A.; Havell, Edward A.; Horton, John R.; Russell, Perry W.; Orndorff, Paul E.
2000-01-01
Five Escherichia coli type 1 pilus mutants that had point mutations in fimH, the gene encoding the type 1 pilus adhesin FimH, were characterized. FimH is a minor component of type 1 pili that is required for the pili to bind and agglutinate guinea pig erythrocytes in a mannose-inhibitable manner. Point mutations were located by DNA sequencing and deletion mapping. All mutations mapped within the signal sequence or in the first 28% of the predicted mature protein. All mutations were missense mutations except for one, a frameshift lesion that was predicted to cause the loss of approximately 60% of the mature FimH protein. Bacterial agglutination tests with polyclonal antiserum raised to a LacZ-FimH fusion protein failed to confirm that parental amounts of FimH cross-reacting material were expressed in four of the five mutants. The remaining mutant, a temperature-sensitive (ts) fimH mutant that agglutinated guinea pig erythrocytes after growth at 31°C but not at 42°C, reacted with antiserum at both temperatures in a manner similar to the parent. Consequently, this mutant was chosen for further study. Temperature shift experiments revealed that new FimH biosynthesis was required for the phenotypic change. Guinea pig erythrocyte and mouse macrophage binding experiments using the ts mutant grown at the restrictive and permissive temperatures revealed that whereas erythrocyte binding was reduced to a level comparable to that of a fimH insertion mutant at the restrictive temperature, mouse peritoneal macrophages were bound with parental efficiency at both the permissive and restrictive temperatures. Also, macrophage binding by the ts mutant was insensitive to mannose inhibition after growth at 42°C but sensitive after growth at 31°C. The ts mutant thus binds macrophages with one receptor specificity at 31°C and another at 42°C. PMID:10869080
-
Rational design of aminoacyl-tRNA synthetase specific for p-acetyl-L-phenylalanine.
Sun, Renhua; Zheng, Heng; Fang, Zhengzhi; Yao, Wenbing
2010-01-01
The Methanococcus jannaschii tRNA(Tyr)/tyrosyl-tRNA synthetase pair has been engineered to incorporate unnatural amino acids into proteins in Escherichia coli site-specifically. In order to add other unnatural amino acids into proteins by this approach, the amino acid binding site of M. jannaschii tyrosyl-tRNA synthetase need to be mutated. The crystal structures of M. jannaschii tyrosyl-tRNA synthetase and its mutations were determined, which provided an opportunity to design aminoacyl-tRNA synthetases specific for other unnatural amino acids. In our study, we attempted to design aminoacyl-tRNA synthetases being able to deliver p-acetyl-L-phenylalanine into proteins. p-Acetyl-L-phenylalanine was superimposed on tyrosyl in M. jannaschii tyrosyl-tRNA synthetase-tyrosine complex. Tyr32 needed to be changed to non-polar amino acid with shorter side chain, Val, Leu, Ile, Gly or Ala, in order to reduce steric clash and provide hydrophobic environment to acetyl on p-acetyl-L-phenylalanine. Asp158 and Ile159 would be changed to specific amino acids for the same reason. So we designed 60 aminoacyl-tRNA synthetases. Binding of these aminoacyl-tRNA synthetases with p-acetyl-L-phenylalanine indicated that only 15 of them turned out to be able to bind p-acetyl-L-phenylalanine with reasonable poses. Binding affinity computation proved that the mutation of Tyr32Leu and Asp158Gly benefited p-acetyl-L-phenylalanine binding. And two of the designed aminoacyl-tRNA synthetases had considerable binding affinities. They seemed to be very promising to be able to incorporate p-acetyl-L-phenylalanine into proteins in E. coli. The results show that the combination of homology modeling and molecular docking is a feasible method to filter inappropriate mutations in molecular design and point out beneficial mutations. Copyright 2009 Elsevier Inc. All rights reserved.
-
Das, Payel; Li, Jingyuan; Royyuru, Ajay K; Zhou, Ruhong
2009-08-01
Historically, influenza pandemics have been triggered when an avian influenza virus or a human/avian reassorted virus acquires the ability to replicate efficiently and become transmissible in the human population. Most critically, the major surface glycoprotein hemagglutinin (HA) must adapt to the usage of human-like (alpha-2,6-linked) sialylated glycan receptors. Therefore, identification of mutations that can switch the currently circulating H5N1 HA receptor binding specificity from avian to human might provide leads to the emergence of pandemic H5N1 viruses. To define such mutations in the H5 subtype, here we provide a computational framework that combines molecular modeling with extensive free energy simulations. Our results show that the simulated binding affinities are in good agreement with currently available experimental data. Moreover, we predict that one double mutation (V135S and A138S) in HA significantly enhances alpha-2,6-linked receptor recognition by the H5 subtype. Our simulations indicate that this double mutation in H5N1 HA increases the binding affinity to alpha-2,6-linked sialic acid receptors by 2.6 +/- 0.7 kcal/mol per HA monomer that primarily arises from the electrostatic interactions. Further analyses reveal that introduction of this double mutation results in a conformational change in the receptor binding pocket of H5N1 HA. As a result, a major rearrangement occurs in the hydrogen-bonding network of HA with the human receptor, making the human receptor binding pattern of double mutant H5N1 HA surprisingly similar to that observed in human H1N1 HA. These large scale molecular simulations on single and double mutants thus provide new insights into our understanding toward human adaptation of the avian H5N1 virus. 2009 Wiley Periodicals, Inc.
-
Yanagawa, Yoshimaro; Aoki, Tomoyuki; Morimura, Tadashi; Araki, Osamu; Kimura, Takao; Ogiwara, Takayuki; Kotajima, Nobuo; Yanagawa, Masumi; Murakami, Masami
2014-01-01
In Japanese pediatric patients with thyrotropin (TSH) resistance, the R450H mutation in TSH receptor gene (TSHR) is occasionally observed. We studied the frequency and clinical implication of the R450H mutation in TSHR in the general population of Japanese adults using smart amplification process 2 (SmartAmp2). We designed SmartAmp2 primer sets to detect this mutation using a drop of whole blood. We analyzed thyroid function, antithyroid antibodies, and this mutation in 429 Japanese participants who had not been found to have thyroid disease. Two cases without antithyroid antibodies were heterozygous for the R450H mutation in TSHR. Thus, the prevalence of this mutation was 0.47% in the general population and 0.63% among those without antithyroid antibodies. Their serum TSH concentrations were higher than the average TSH concentration not only in subjects without antithyroid antibodies but also in those with antithyroid antibodies. The R450H mutation in TSHR is relatively common in the Japanese population and potentially affects thyroid function. The present study demonstrates that the SmartAmp2 method is useful to detect the R450H mutation in TSHR, which is one of the common causes of TSH resistance in the Japanese population. PMID:24895636
-
Cardoso, C; Lutz, Y; Mignon, C; Compe, E; Depetris, D; Mattei, M G; Fontes, M; Colleaux, L
2000-10-01
Mutations in the XNP/ATR-X gene, located in Xq13.3, are associated with several X linked mental retardation syndromes, the best known being alpha thalassaemia with mental retardation (ATR-X). The XNP/ATR-X protein belongs to the family of SWI/SNF DNA helicases and contains three C2-C2 type zinc fingers of unknown function. Previous studies have shown that 65% of mutations of XNP have been found within the zinc finger domain (encoded by exons 7, 8, and the beginning of exon 9) while 35% of the mutations have been found in the helicase domain extending over 3 kb at the C-terminus of the protein. Although different types of mutations have been identified, no specific genotype-phenotype correlation has been found, suggesting that gene alteration leads to a loss of function irrespective of mutation type. Our aims were to understand the function of the XNP/ATR-X protein better, with specific attention to the functional consequences of mutations to the zinc finger domain. We used monoclonal antibodies directed against the XNP/ATR-X protein and performed immunocytochemical and western blot analyses, which showed altered or absent XNP/ATR-X expression in cells of affected patients. In addition, we used in vitro experiments to show that the zinc finger domain can mediate double stranded DNA binding and found that the DNA binding capacity of mutant forms in ATR-X patients is severely reduced. These data provide insights into the understanding of the functional significance of XNP/ATR-X mutations.
-
Valdramidou, Dimitra; Humphries, Martin J.; Mould, A. Paul
2012-01-01
Integrin-ligand interactions are regulated in a complex manner by divalent cations, and previous studies have identified ligand-competent, stimulatory, and inhibitory cation-binding sites. In collagen-binding integrins, such as α2β1, ligand recognition takes place exclusively at the α subunit I domain. However, activation of the αI domain depends on its interaction with a structurally similar domain in the β subunit known as the I-like or βI domain. The top face of the βI domain contains three cation-binding sites: the metal-ion dependent adhesion site (MIDAS), the ADMIDAS (adjacent to MIDAS) and LIMBS (ligand-associated metal binding site). The role of these sites in controlling ligand binding to the αI domain has yet to be elucidated. Mutation of the MIDAS or LIMBS completely blocked collagen binding to α2β1; in contrast mutation of the ADMIDAS reduced ligand recognition but this effect could be overcome by the activating mAb TS2/16. Hence, the MIDAS and LIMBS appear to be essential for the interaction between αI and βI whereas occupancy of the ADMIDAS has an allosteric effect on the conformation of βI. An activating mutation in the α2 I domain partially restored ligand binding to the MIDAS and LIMBS mutants. Analysis of the effects of Ca2+, Mg2+ and Mn2+ on ligand binding to these mutants showed that the MIDAS is a ligand-competent site through which Mn2+ stimulates ligand binding, whereas the LIMBS is a stimulatory Ca2+-binding site, occupancy of which increases the affinity of Mg2+ for the MIDAS. PMID:18820259
-
Comprehensive mutational profiling of core binding factor acute myeloid leukemia
Duployez, Nicolas; Marceau-Renaut, Alice; Boissel, Nicolas; Petit, Arnaud; Bucci, Maxime; Geffroy, Sandrine; Lapillonne, Hélène; Renneville, Aline; Ragu, Christine; Figeac, Martin; Celli-Lebras, Karine; Lacombe, Catherine; Micol, Jean-Baptiste; Abdel-Wahab, Omar; Cornillet, Pascale; Ifrah, Norbert; Dombret, Hervé; Leverger, Guy; Jourdan, Eric
2016-01-01
Acute myeloid leukemia (AML) with t(8;21) or inv(16) have been recognized as unique entities within AML and are usually reported together as core binding factor AML (CBF-AML). However, there is considerable clinical and biological heterogeneity within this group of diseases, and relapse incidence reaches up to 40%. Moreover, translocations involving CBFs are not sufficient to induce AML on its own and the full spectrum of mutations coexisting with CBF translocations has not been elucidated. To address these issues, we performed extensive mutational analysis by high-throughput sequencing in 215 patients with CBF-AML enrolled in the Phase 3 Trial of Systematic Versus Response-adapted Timed-Sequential Induction in Patients With Core Binding Factor Acute Myeloid Leukemia and Treating Patients with Childhood Acute Myeloid Leukemia with Interleukin-2 trials (age, 1-60 years). Mutations in genes activating tyrosine kinase signaling (including KIT, N/KRAS, and FLT3) were frequent in both subtypes of CBF-AML. In contrast, mutations in genes that regulate chromatin conformation or encode members of the cohesin complex were observed with high frequencies in t(8;21) AML (42% and 18%, respectively), whereas they were nearly absent in inv(16) AML. High KIT mutant allele ratios defined a group of t(8;21) AML patients with poor prognosis, whereas high N/KRAS mutant allele ratios were associated with the lack of KIT or FLT3 mutations and a favorable outcome. In addition, mutations in epigenetic modifying or cohesin genes were associated with a poor prognosis in patients with tyrosine kinase pathway mutations, suggesting synergic cooperation between these events. These data suggest that diverse cooperating mutations may influence CBF-AML pathophysiology as well as clinical behavior and point to potential unique pathogenesis of t(8;21) vs inv(16) AML. PMID:26980726
-
Demir, Hande; Donner, Iikki; Kivipelto, Leena; Kuismin, Outi; Schalin-Jäntti, Camilla; De Menis, Ernesto; Karhu, Auli
2014-01-01
Pituitary adenomas are neoplasms of the anterior pituitary lobe and account for 15-20% of all intracranial tumors. Although most pituitary tumors are benign they can cause severe symptoms related to tumor size as well as hypopituitarism and/or hypersecretion of one or more pituitary hormones. Most pituitary adenomas are sporadic, but it has been estimated that 5% of patients have a familial background. Germline mutations of the tumor suppressor gene aryl hydrocarbon receptor-interacting protein (AIP) predispose to hereditary pituitary neoplasia. Recently, it has been demonstrated that AIP mutations predispose to pituitary tumorigenesis through defective inhibitory GTP binding protein (Gαi) signaling. This finding prompted us to examine whether germline loss-of-function mutations in inhibitory guanine nucleotide (GTP) binding protein alpha (GNAI) loci are involved in genetic predisposition of pituitary tumors. To our knowledge, this is the first time GNAI genes are sequenced in order to examine the occurrence of inactivating germline mutations. Thus far, only somatic gain-of-function hot-spot mutations have been studied in these loci. Here, we have analyzed the coding regions of GNAI1, GNAI2, and GNAI3 in a set of young sporadic somatotropinoma patients (n = 32; mean age of diagnosis 32 years) and familial index cases (n = 14), thus in patients with a disease phenotype similar to that observed in AIP mutation carriers. In addition, expression of Gαi proteins was studied in human growth hormone (GH), prolactin (PRL), adrenocorticotropic hormone (ACTH)-secreting and non-functional pituitary tumors. No pathogenic germline mutations affecting the Gαi proteins were detected. The result suggests that loss-of-function mutations of GNAI loci are rare or nonexistent in familial pituitary adenomas.
-
Demir, Hande; Donner, Iikki; Kivipelto, Leena; Kuismin, Outi; Schalin-Jäntti, Camilla; De Menis, Ernesto; Karhu, Auli
2014-01-01
Pituitary adenomas are neoplasms of the anterior pituitary lobe and account for 15–20% of all intracranial tumors. Although most pituitary tumors are benign they can cause severe symptoms related to tumor size as well as hypopituitarism and/or hypersecretion of one or more pituitary hormones. Most pituitary adenomas are sporadic, but it has been estimated that 5% of patients have a familial background. Germline mutations of the tumor suppressor gene aryl hydrocarbon receptor-interacting protein (AIP) predispose to hereditary pituitary neoplasia. Recently, it has been demonstrated that AIP mutations predispose to pituitary tumorigenesis through defective inhibitory GTP binding protein (Gαi) signaling. This finding prompted us to examine whether germline loss-of-function mutations in inhibitory guanine nucleotide (GTP) binding protein alpha (GNAI) loci are involved in genetic predisposition of pituitary tumors. To our knowledge, this is the first time GNAI genes are sequenced in order to examine the occurrence of inactivating germline mutations. Thus far, only somatic gain-of-function hot-spot mutations have been studied in these loci. Here, we have analyzed the coding regions of GNAI1 , GNAI2, and GNAI3 in a set of young sporadic somatotropinoma patients (n = 32; mean age of diagnosis 32 years) and familial index cases (n = 14), thus in patients with a disease phenotype similar to that observed in AIP mutation carriers. In addition, expression of Gαi proteins was studied in human growth hormone (GH), prolactin (PRL), adrenocorticotropic hormone (ACTH)-secreting and non-functional pituitary tumors. No pathogenic germline mutations affecting the Gαi proteins were detected. The result suggests that loss-of-function mutations of GNAI loci are rare or nonexistent in familial pituitary adenomas. PMID:25291362
-
P., Sneha; D., Thirumal Kumar; C., George Priya Doss; R., Siva; Zayed, Hatem
2017-01-01
Maturity-onset diabetes of the young type 3 (MODY3) is a non-ketotic form of diabetes associated with poor insulin secretion. Over the past years, several studies have reported the association of missense mutations in the Hepatocyte Nuclear Factor 1 Alpha (HNF1A) with MODY3. Missense mutations in the POU homeodomain (POUH) of HNF1A hinder binding to the DNA, thereby leading to a dysfunctional protein. Missense mutations of the HNF1A were retrieved from public databases and subjected to a three-step computational mutational analysis to identify the underlying mechanism. First, the pathogenicity and stability of the mutations were analyzed to determine whether they alter protein structure and function. Second, the sequence conservation and DNA-binding sites of the mutant positions were assessed; as HNF1A protein is a transcription factor. Finally, the biochemical properties of the biological system were validated using molecular dynamic simulations in Gromacs 4.6.3 package. Two arginine residues (131 and 203) in the HNF1A protein are highly conserved residues and contribute to the function of the protein. Furthermore, the R131W, R131Q, and R203C mutations were predicted to be highly deleterious by in silico tools and showed lower binding affinity with DNA when compared to the native protein using the molecular docking analysis. Triplicate runs of molecular dynamic (MD) simulations (50ns) revealed smaller changes in patterns of deviation, fluctuation, and compactness, in complexes containing the R131Q and R131W mutations, compared to complexes containing the R203C mutant complex. We observed reduction in the number of intermolecular hydrogen bonds, compactness, and electrostatic potential, as well as the loss of salt bridges, in the R203C mutant complex. Substitution of arginine with cysteine at position 203 decreases the affinity of the protein for DNA, thereby destabilizing the protein. Based on our current findings, the MD approach is an important tool for elucidating the impact and affinity of mutations in DNA-protein interactions and understanding their function. PMID:28410371
-
NASA Astrophysics Data System (ADS)
Singh, Warispreet; Karabencheva-Christova, Tatyana G.; Black, Gary W.; Ainsley, Jon; Dover, Lynn; Christov, Christo Z.
2016-01-01
Heme d1, a vital tetrapyrrol involved in the denitrification processes is synthesized from its precursor molecule precorrin-2 in a chemical reaction catalysed by an S-adenosyl-L-methionine (SAM) dependent Methyltransferase (NirE). The NirE enzyme catalyses the transfer of a methyl group from the SAM to uroporphyrinogen III and serves as a novel potential drug target for the pharmaceutical industry. An important insight into the structure-activity relationships of NirE has been revealed by elucidating its crystal structure, but there is still no understanding about how conformational flexibility influences structure, cofactor and substrate binding by the enzyme as well as the structural effects of mutations of residues involved in binding and catalysis. In order to provide this missing but very important information we performed a comprehensive atomistic molecular dynamics study which revealed that i) the binding of the substrate contributes to the stabilization of the structure of the full complex; ii) conformational changes influence the orientation of the pyrrole rings in the substrate, iii) more open conformation of enzyme active site to accommodate the substrate as an outcome of conformational motions; and iv) the mutations of binding and active site residues lead to sensitive structural changes which influence binding and catalysis.
-
Siebler, Hollie M.; Lada, Artem G.; Baranovskiy, Andrey G.; Tahirov, Tahir H.; Pavlov, Youri I.
2014-01-01
Unrepaired DNA lesions often stall replicative DNA polymerases and are bypassed by translesion synthesis (TLS) to prevent replication fork collapse. Mechanisms of TLS are lesion- and species-specific, with a prominent role of specialized DNA polymerases with relaxed active sites. After nucleotide(s) are incorporated across from the altered base(s), the aberrant primer termini are typically extended by DNA polymerase ζ (pol ζ). As a result, pol ζ is responsible for most DNA damage-induced mutations. The mechanisms of sequential DNA polymerase switches in vivo remain unclear. The major replicative DNA polymerase δ (pol δ) shares two accessory subunits, called Pol31/Pol32 in yeast, with pol ζ. Inclusion of Pol31/Pol32 in the pol δ/pol ζ holoenzymes requires a [4Fe–4S] cluster in C-termini of the catalytic subunits. Disruption of this cluster in Pol ζ or deletion of POL32 attenuates induced mutagenesis. Here we describe a novel mutation affecting the catalytic subunit of pol ζ, rev3ΔC, which provides insight into the regulation of pol switches. Strains with Rev3ΔC, lacking the entire C-terminal domain and therefore the platform for Pol31/Pol32 binding, are partially proficient in Pol32-dependent UV-induced mutagenesis. This suggests an additional role of Pol32 in TLS, beyond being a pol ζ subunit, related to pol δ. In search for members of this regulatory pathway, we examined the effects of Maintenance of Genome Stability 1 (Mgs1) protein on mutagenesis in the absence of Rev3–Pol31/Pol32 interaction. Mgs1 may compete with Pol32 for binding to PCNA. Mgs1 overproduction suppresses induced mutagenesis, but had no effect on UV-mutagenesis in the rev3ΔC strain, suggesting that Mgs1 exerts its inhibitory effect by acting specifically on Pol32 bound to pol ζ. The evidence for differential regulation of Pol32 in pol δ and pol ζ emphasizes the complexity of polymerase switches. PMID:24819597
-
Kiani, Farooq; Schwarzl, Sonja; Fischer, Stefan; Efferth, Thomas
2007-07-18
Loss of function of dimeric glucose-6-phosphate dehydrogenase (G6PD) represents the most common inborn error of metabolism throughout the world affecting an estimated 400 million people. In Germany, this enzymopathy is very rare. On the basis of G6PD crystal structures, we have analyzed six G6PD variants of German ancestry by three-dimensional modeling. All mutations present in the German population are either close to one of the three G6P or NADP(+) units or to the interface of the two monomers. Two of the three mutated amino acids of G6PD Vancouver are closer to the binding site of NADP(+). The G6PD Aachen mutation is also closer to the second NADP(+) unit. The G6PD Wayne mutation is closer to the G6P binding region. These mutations may affect the binding of G6P and NADP(+) units. Three mutations, i.e. G6PD Munich, G6PD Riverside and G6PD Gastonia, lie closer to the interface of the two monomers. These may also affect the interface of two monomers. None of these G6PD variants share mutations with the common G6PD variants known from the Mediterranean, Near East, or Africa indicating that they have developed independently. The G6PD variants have been compared with mutants from other populations and the implications for survival of G6PD variants from natural selection have been discussed.
-
Sun, Huiyong; Li, Youyong; Tian, Sheng; Wang, Junmei; Hou, Tingjun
2014-01-01
Tyrosine kinases are regarded as excellent targets for chemical drug therapy of carcinomas. However, under strong purifying selection, drug resistance usually occurs in the cancer cells within a short term. Many cases of drug resistance have been found to be associated with secondary mutations in drug target, which lead to the attenuated drug-target interactions. For example, recently, an acquired secondary mutation, G2032R, has been detected in the drug target, ROS1 tyrosine kinase, from a crizotinib-resistant patient, who responded poorly to crizotinib within a very short therapeutic term. It was supposed that the mutation was located at the solvent front and might hinder the drug binding. However, a different fact could be uncovered by the simulations reported in this study. Here, free energy surfaces were characterized by the drug-target distance and the phosphate-binding loop (P-loop) conformational change of the crizotinib-ROS1 complex through advanced molecular dynamics techniques, and it was revealed that the more rigid P-loop region in the G2032R-mutated ROS1 was primarily responsible for the crizotinib resistance, which on one hand, impaired the binding of crizotinib directly, and on the other hand, shortened the residence time induced by the flattened free energy surface. Therefore, both of the binding affinity and the drug residence time should be emphasized in rational drug design to overcome the kinase resistance. PMID:25033171
-
Sun, Huiyong; Li, Youyong; Tian, Sheng; Wang, Junmei; Hou, Tingjun
2014-07-01
Tyrosine kinases are regarded as excellent targets for chemical drug therapy of carcinomas. However, under strong purifying selection, drug resistance usually occurs in the cancer cells within a short term. Many cases of drug resistance have been found to be associated with secondary mutations in drug target, which lead to the attenuated drug-target interactions. For example, recently, an acquired secondary mutation, G2032R, has been detected in the drug target, ROS1 tyrosine kinase, from a crizotinib-resistant patient, who responded poorly to crizotinib within a very short therapeutic term. It was supposed that the mutation was located at the solvent front and might hinder the drug binding. However, a different fact could be uncovered by the simulations reported in this study. Here, free energy surfaces were characterized by the drug-target distance and the phosphate-binding loop (P-loop) conformational change of the crizotinib-ROS1 complex through advanced molecular dynamics techniques, and it was revealed that the more rigid P-loop region in the G2032R-mutated ROS1 was primarily responsible for the crizotinib resistance, which on one hand, impaired the binding of crizotinib directly, and on the other hand, shortened the residence time induced by the flattened free energy surface. Therefore, both of the binding affinity and the drug residence time should be emphasized in rational drug design to overcome the kinase resistance.
-
Development of Pineapple Microsatellite Markers and Germplasm Genetic Diversity Analysis
Tong, Helin; Chen, You; Wang, Jingyi; Chen, Yeyuan; Sun, Guangming; He, Junhu; Wu, Yaoting
2013-01-01
Two methods were used to develop pineapple microsatellite markers. Genomic library-based SSR development: using selectively amplified microsatellite assay, 86 sequences were generated from pineapple genomic library. 91 (96.8%) of the 94 Simple Sequence Repeat (SSR) loci were dinucleotide repeats (39 AC/GT repeats and 52 GA/TC repeats, accounting for 42.9% and 57.1%, resp.), and the other three were mononucleotide repeats. Thirty-six pairs of SSR primers were designed; 24 of them generated clear bands of expected sizes, and 13 of them showed polymorphism. EST-based SSR development: 5659 pineapple EST sequences obtained from NCBI were analyzed; among 1397 nonredundant EST sequences, 843 were found containing 1110 SSR loci (217 of them contained more than one SSR locus). Frequency of SSRs in pineapple EST sequences is 1SSR/3.73 kb, and 44 types were found. Mononucleotide, dinucleotide, and trinucleotide repeats dominate, accounting for 95.6% in total. AG/CT and AGC/GCT were the dominant type of dinucleotide and trinucleotide repeats, accounting for 83.5% and 24.1%, respectively. Thirty pairs of primers were designed for each of randomly selected 30 sequences; 26 of them generated clear and reproducible bands, and 22 of them showed polymorphism. Eighteen pairs of primers obtained by the one or the other of the two methods above that showed polymorphism were selected to carry out germplasm genetic diversity analysis for 48 breeds of pineapple; similarity coefficients of these breeds were between 0.59 and 1.00, and they can be divided into four groups accordingly. Amplification products of five SSR markers were extracted and sequenced, corresponding repeat loci were found and locus mutations are mainly in copy number of repeats and base mutations in the flanking region. PMID:24024187
-
Inaccurate DNA synthesis in cell extracts of yeast producing active human DNA polymerase iota.
Makarova, Alena V; Grabow, Corinn; Gening, Leonid V; Tarantul, Vyacheslav Z; Tahirov, Tahir H; Bessho, Tadayoshi; Pavlov, Youri I
2011-01-31
Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn(2+) ions, can bypass some DNA lesions and misincorporates "G" opposite template "T" more frequently than incorporates the correct "A." We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of "G" versus "A" method of Gening, abbreviated as "misGvA"). We provide unambiguous proof of the "misGvA" approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The "misGvA" activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts.
-
Andera, L; Spangler, C J; Galeone, A; Mayol, L; Geiduschek, E P
1994-02-11
TF1, a homodimeric DNA-binding and -bending protein with a preference for hydroxymethyluracil-containing DNA is the Bacillus subtilis-encoded homolog of the bacterial HU proteins and of the E. coli integration host factor. A temperature-sensitive mutation at amino acid 25 of TF1 (L25-->A) and two intragenic second site revertants at amino acids 15 (E15-->G) and 32 (L32-->I) were previously identified and their effects on virus development were examined. The DNA-binding properties of these proteins and the thermal stability of their secondary structures have now been analyzed. Amino acids 15 and 32 are far removed from the putative DNA-binding domains of TF1 but changes there exert striking effects on DNA affinity that correlate with effects on structure. The double mutant protein TF1-G15I32 binds to a preferred site in hydroxymethyluracil-containing DNA 40 times more tightly, denatures at higher temperature (delta tm = 21 degrees C), and also exchanges subunits much more slowly than does the wild-type protein. The L25-->A mutation makes TF1 secondary structure and DNA-binding highly salt concentration-dependent. The E15-->G mutation partly suppresses this effect: secondary structure of TF1-A25G15 is restored at 21 degrees C by 1 M NaCl or, at low NaCl concentration, by binding to DNA.
-
Baek, Seung Cheol; Ho, Thien-Hoang; Lee, Hyun Woo; Jung, Won Kyeong; Gang, Hyo-Seung; Kang, Lin-Woo; Kim, Hoon
2017-05-01
β-1,3-1,4-Glucanase (BGlc8H) from Paenibacillus sp. X4 was mutated by error-prone PCR or truncated using termination primers to improve its enzyme properties. The crystal structure of BGlc8H was determined at a resolution of 1.8 Å to study the possible roles of mutated residues and truncated regions of the enzyme. In mutation experiments, three clones of EP 2-6, 2-10, and 5-28 were finally selected that exhibited higher specific activities than the wild type when measured using their crude extracts. Enzyme variants of BG 2-6 , BG 2-10 , and BG 5-28 were mutated at two, two, and six amino acid residues, respectively. These enzymes were purified homogeneously by Hi-Trap Q and CHT-II chromatography. Specific activity of BG 5-28 was 2.11-fold higher than that of wild-type BG wt , whereas those of BG 2-6 and BG 2-10 were 0.93- and 1.19-fold that of the wild type, respectively. The optimum pH values and temperatures of the variants were nearly the same as those of BG wt (pH 5.0 and 40 °C, respectively). However, the half-life of the enzyme activity and catalytic efficiency (k cat /K m ) of BG 5-28 were 1.92- and 2.12-fold greater than those of BG wt at 40 °C, respectively. The catalytic efficiency of BG 5-28 increased to 3.09-fold that of BG wt at 60 °C. These increases in the thermostability and catalytic efficiency of BG 5-28 might be useful for the hydrolysis of β-glucans to produce fermentable sugars. Of the six mutated residues of BG 5-28 , five residues were present in mature BGlc8H protein, and two of them were located in the core scaffold of BGlc8H and the remaining three residues were in the substrate-binding pocket forming loop regions. In truncation experiments, three forms of C-terminal truncated BGlc8H were made, which comprised 360, 286, and 215 amino acid residues instead of the 409 residues of the wild type. No enzyme activity was observed for these truncated enzymes, suggesting the complete scaffold of the α 6 /α 6 -double-barrel structure is essential for enzyme activity.
-
Engineering cofactor and ligand binding in an artificial neuroglobin
NASA Astrophysics Data System (ADS)
Zhang, Lei
HP-7 is one artificial mutated oxygen transport protein, which operates via a mechanism akin to human neuroglobin and cytoglobin. This protein destabilizes one of two heme-ligating histidine residues by coupling histidine side chain ligation with the burial of three charged glutamate residues on the same helix. Replacement of these glutamate residues with alanine, which has a neutral hydrophobicity, slows gaseous ligand binding 22-fold, increases the affinity of the distal histidine ligand by a factor of thirteen, and decreases the binding affinity of carbon monoxide, a nonreactive oxygen analogue, three-fold. Paradoxically, it also decreases heme binding affinity by a factor of three in the reduced state and six in the oxidized state. Application of a two-state binding model, in which an initial pentacoordinate binding event is followed by a protein conformational change to hexacoordinate, provides insight into the mechanism of this seemingly counterintuitive result: the initial pentacoordinate encounter complex is significantly destabilized by the loss of the glutamate side chains, and the increased affinity for the distal histidine only partially compensates. These results point to the importance of considering each oxidation and conformational state in the design of functional artificial proteins. We have also examined the effects these mutations have on function. The K d of the nonnreactive oxygen analogue carbon monoxide (CO) is only decreased three-fold, despite the large increase in distal histidine affinity engendered by the 22-fold decrease in the histidine ligand off-rate. This is a result of the four-fold increase in affinity for CO binding to the pentacoordinate state. Oxygen binds to HP7 with a Kd of 117 µM, while the mutant rapidly oxidizes when exposed to oxygen. EPR analysis of both ferric hemoproteins demonstrates that the mutation increases disorder at the heme binding site. NMR-detected deuterium exchange demonstrates that the mutation causes a large increase in water penetration into the protein core. The inability of the mutant protein may thus either be due to increased water penetration, the large decrease in binding rate caused by the increase in distal histidine affinity, or a combination of the two factors.
-
Wang, Lingyun; Yan, Feng
2017-12-09
Heterogeneous nuclear ribonucleoprotein F (hnRNP F) controls the expression of various genes through regulating the alternative splicing of pre-mRNAs in the nucleus. It uses three quasi-RNA recognition motifs (qRRMs) to recognize G-tract RNA which contains at least three consecutive guanines. The structures containing qRRMs of hnRNP F in complex with G-tract RNA have been determined by nuclear magnetic resonance (NMR) spectroscopy, shedding light on the recognition mechanism of qRRMs with G-tract RNA. However, knowledge of the recognition details is still lacking. To investigate how qRRMs specifically bind with G-tract RNA and how the mutations of any guanine to an adenine in the G-tract affect the binding, molecular dynamics simulations with binding free energy analysis were performed based on the NMR structure of qRRM2 in complex with G-tract RNA. Simulation results demonstrate that qRRM2 binds strongly with G-tract RNA, but any mutation of the G-tract leads to a drastic reduction of the binding free energy. Further comparisons of the energetic components reveal that van der Waals and non-polar interactions play essential roles in the binding between qRRM2 and G-tract RNA, but the interactions are weakened by the effect of RNA mutations. Structural and dynamical analyses indicate that when qRRM2 binds with G-tract RNA, both qRRM2 and G-tract maintain stabilized structures and dynamics; however, the stability is disrupted by the mutations of the G-tract. These results provide novel insights into the recognition mechanism of qRRM2 with G-tract RNA that are not elucidated by the NMR technique. Copyright © 2017 Elsevier Inc. All rights reserved.
-
Vargiu, Attilio V; Collu, Francesca; Schulz, Robert; Pos, Klaas M; Zacharias, Martin; Kleinekathöfer, Ulrich; Ruggerone, Paolo
2011-07-20
The tripartite efflux pump AcrAB-TolC is responsible for the intrinsic and acquired multidrug resistance in Escherichia coli. Its active part, the homotrimeric transporter AcrB, is in charge of the selective binding of substrates and energy transduction. The mutation F610A has been shown to significantly reduce the minimum inhibitory concentration of doxorubicin and many other substrates, although F610 does not appear to interact strongly with them. Biochemical study of transport kinetics in AcrB is not yet possible, except for some β-lactams, and other techniques should supply this important information. Therefore, in this work, we assess the impact of the F610A mutation on the functionality of AcrB by means of computational techniques, using doxorubicin as substrate. We found that the compound slides deeply inside the binding pocket after mutation, increasing the strength of the interaction. During subsequent conformational alterations of the transporter, doxorubicin was either not extruded from the binding site or displaced along a direction other than the one associated with extrusion. Our study indicates how subtle interactions determine the functionality of multidrug transporters, since decreased transport might not be simplistically correlated to decreased substrate binding affinity.
-
Ni, Duan; Song, Kun; Zhang, Jian; Lu, Shaoyong
2017-10-26
Ras proteins, as small GTPases, mediate cell proliferation, survival and differentiation. Ras mutations have been associated with a broad spectrum of human cancers and thus targeting Ras represents a potential way forward for cancer therapy. A recently reported monobody NS1 allosterically disrupts the Ras-mediated signaling pathway, but its efficacy is reduced by R135K mutation in H-Ras. However, the detailed mechanism is unresolved. Here, using molecular dynamics (MD) simulations and dynamic network analysis, we explored the molecular mechanism for the unbinding of NS1 to H-Ras and shed light on the underlying allosteric network in H-Ras. MD simulations revealed that the overall structures of the two complexes did not change significantly, but the H-Ras-NS1 interface underwent significant conformational alteration in the mutant Binding free energy analysis showed that NS1 binding was unfavored after R135K mutation, which resulted in the unfavorable binding of NS1. Furthermore, the critical residues on H-Ras responsible for the loss of binding of NS1 were identified. Importantly, the allosteric networks for these important residues were revealed, which yielded a novel insight into the allosteric regulatory mechanism of H-Ras.
-
Song, Kun; Zhang, Jian; Lu, Shaoyong
2017-01-01
Ras proteins, as small GTPases, mediate cell proliferation, survival and differentiation. Ras mutations have been associated with a broad spectrum of human cancers and thus targeting Ras represents a potential way forward for cancer therapy. A recently reported monobody NS1 allosterically disrupts the Ras-mediated signaling pathway, but its efficacy is reduced by R135K mutation in H-Ras. However, the detailed mechanism is unresolved. Here, using molecular dynamics (MD) simulations and dynamic network analysis, we explored the molecular mechanism for the unbinding of NS1 to H-Ras and shed light on the underlying allosteric network in H-Ras. MD simulations revealed that the overall structures of the two complexes did not change significantly, but the H-Ras–NS1 interface underwent significant conformational alteration in the mutant Binding free energy analysis showed that NS1 binding was unfavored after R135K mutation, which resulted in the unfavorable binding of NS1. Furthermore, the critical residues on H-Ras responsible for the loss of binding of NS1 were identified. Importantly, the allosteric networks for these important residues were revealed, which yielded a novel insight into the allosteric regulatory mechanism of H-Ras. PMID:29072601
-
A Meier-Gorlin syndrome mutation impairs the ORC1-nucleosome association.
Zhang, Wei; Sankaran, Saumya; Gozani, Or; Song, Jikui
2015-05-15
Recent studies have identified several genetic mutations within the BAH domain of human Origin Recognition Complex subunit 1 (hORC1BAH), including the R105Q mutation, implicated in Meier-Gorlin Syndrome (MGS). However, the pathological role of the hORC1 R105Q mutation remains unclear. In this study, we have investigated the interactions of the hORC1BAH domain with histone H4K20me2, DNA, and the nucleosome core particle labeled with H4Kc20me2, a chemical analog of H4K20me2. Our study revealed a nucleosomal DNA binding site for hORC1BAH. The R105Q mutation reduces the hORC1BAH-DNA binding affinity, leading to impaired hORC1BAH-nucleosome interaction, which likely influences DNA replication initiation and MGS pathogenesis. This study provides an etiologic link between the hORC1 R105Q mutation and MGS.
-
Chimeric protein identification of dystrophic, Pierson and other laminin polymerization residues
McKee, Karen K.; Aleksandrova, Maya; Yurchenco, Peter D.
2018-01-01
Laminin polymerization is a key step of basement membrane self-assembly that depends on the binding of the three different N-terminal globular LN domains. Several mutations in the LN domains cause LAMA2-deficient muscular dystrophy and LAMB2-deficient Pierson syndrome. These mutations may affect polymerization. A novel approach to identify the amino acid residues required for polymerization has been applied to an analysis of these and other laminin LN mutations. The approach utilizes laminin-nidogen chimeric fusion proteins that bind to recombinant non-polymerizing laminins to provide a missing functional LN domain. Single amino acid substitutions introduced into these chimeras were tested to determine if polymerization activity and the ability to assemble on cell surfaces were lost. Several laminin-deficient muscular dystrophy mutations, renal Pierson syndrome mutations, and Drosophila mutations causing defects of heart development were identified as ones causing loss of laminin polymerization. In addition, two novel residues required for polymerization were identified in the laminin γ1 LN domain. PMID:29408412
-
A novel STXBP1 mutation causes typical Rett syndrome in a Japanese girl.
Yuge, Kotaro; Iwama, Kazuhiro; Yonee, Chihiro; Matsufuji, Mayumi; Sano, Nozomi; Saikusa, Tomoko; Yae, Yukako; Yamashita, Yushiro; Mizuguchi, Takeshi; Matsumoto, Naomichi; Matsuishi, Toyojiro
2018-06-01
Rett syndrome (RTT) is a neurodevelopmental disorder mostly caused by mutations in Methyl-CpG-binding protein 2 (MECP2); however, mutations in various other genes may lead to RTT-like phenotypes. Here, we report the first case of a Japanese girl with RTT caused by a novel syntaxin-binding protein 1 (STXBP1) frameshift mutation (c.60delG, p.Lys21Argfs*16). She showed epilepsy at one year of age, regression of acquired psychomotor abilities thereafter, and exhibited stereotypic hand and limb movements at 3 years of age. Her epilepsy onset was earlier than is typical for RTT patients. However, she fully met the 2010 diagnostic criteria of typical RTT. STXBP1 mutations cause early infantile epileptic encephalopathy (EIEE), various intractable epilepsies, and neurodevelopmental disorders. However, the case described here presented a unique clinical presentation of typical RTT without EIEE and a novel STXBP1 mutation. Copyright © 2018 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.
-
Resensitization to Crizotinib by the Lorlatinib ALK Resistance Mutation L1198F
Shaw, Alice T.; Friboulet, Luc; Leshchiner, Ignaty; Gainor, Justin F.; Bergqvist, Simon; Brooun, Alexei; Burke, Benjamin J.; Deng, Ya-Li; Liu, Wei; Dardaei, Leila; Frias, Rosa L.; Schultz, Kate R.; Logan, Jennifer; James, Leonard P.; Smeal, Tod; Timofeevski, Sergei; Katayama, Ryohei; Iafrate, A. John; Le, Long; McTigue, Michele; Getz, Gad
2016-01-01
Summary In a patient who had metastatic anaplastic lymphoma kinase (ALK)-rearranged lung cancer, resistance to crizotinib developed because of a mutation in the ALK kinase domain. This mutation is predicted to result in a substitution of cysteine by tyrosine at amino acid residue 1156 (C1156Y). Her tumor did not respond to a second-generation ALK inhibitor, but it did respond to lorlatinib (PF-06463922), a third-generation inhibitor. When her tumor relapsed, sequencing of the resistant tumor revealed an ALK L1198F mutation in addition to the C1156Y mutation. The L1198F substitution confers resistance to lorlatinib through steric interference with drug binding. However, L1198F paradoxically enhances binding to crizotinib, negating the effect of C1156Y and resensitizing resistant cancers to crizotinib. The patient received crizotinib again, and her cancer-related symptoms and liver failure resolved. PMID:26698910
-
Lena, Anna Maria; Duca, Sara; Novelli, Flavia; Melino, Sonia; Annicchiarico-Petruzzelli, Margherita; Melino, Gerry; Candi, Eleonora
2015-11-13
p63, a member of the p53 family, is a crucial transcription factor for epithelial development and skin homeostasis. Heterozygous mutations in TP63 gene have been associated with human ectodermal dysplasia disorders. Most of these TP63 mutations are missense mutations causing amino acidic substitutions at p63 DNA binding or SAM domains that reduce or abolish the transcriptional activity of mutants p63. A significant number of mutants, however, resides in part of the p63 protein that apparently do not affect DNA binding and/or transcriptional activity, such as the N-terminal domain. Here, we characterize five p63 mutations at the 5' end of TP63 gene aiming to understand the pathogenesis of the diseases and to uncover the role of ΔNp63α N-terminus residues in determining its transactivation potential. Copyright © 2015 Elsevier Inc. All rights reserved.
-
Computational screening and molecular dynamics simulation of disease associated nsSNPs in CENP-E.
Kumar, Ambuj; Purohit, Rituraj
2012-01-01
Aneuploidy and chromosomal instability (CIN) are hallmarks of most solid tumors. Mutations in centroemere proteins have been observed in promoting aneuploidy and tumorigenesis. Recent studies reported that Centromere-associated protein-E (CENP-E) is involved in inducing cancers. In this study we investigated the pathogenic effect of 132 nsSNPs reported in CENP-E using computational platform. Y63H point mutation found to be associated with cancer using SIFT, Polyphen, PhD-SNP, MutPred, CanPredict and Dr. Cancer tools. Further we investigated the binding affinity of ATP molecule to the CENP-E motor domain. Complementarity scores obtained from docking studies showed significant loss in ATP binding affinity of mutant structure. Molecular dynamics simulation was carried to examine the structural consequences of Y63H mutation. Root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (R(g)), solvent accessibility surface area (SASA), energy value, hydrogen bond (NH Bond), eigenvector projection, trace of covariance matrix and atom density analysis results showed notable loss in stability for mutant structure. Y63H mutation was also shown to disrupt the native conformation of ATP binding region in CENP-E motor domain. Docking studies for remaining 18 mutations at 63rd residue position as well as other two computationally predicted disease associated mutations S22L and P69S were also carried to investigate their affect on ATP binding affinity of CENP-E motor domain. Our study provided a promising computational methodology to study the tumorigenic consequences of nsSNPs that have not been characterized and clear clue to the wet lab scientist. Copyright © 2012 Elsevier B.V. All rights reserved.
-
Gatot, Jean-Stéphane; Callebaut, Isabelle; Van Lint, Carine; Demonté, Dominique; Kerkhofs, Pierre; Portetelle, Daniel; Burny, Arsène; Willems, Luc; Kettmann, Richard
2002-01-01
Bovine leukemia virus (BLV) and human T-cell lymphotropic virus type 1 (HTLV-1) belong to the genus of deltaretroviruses. Their entry into the host cell is supposed to be mediated by interactions of the extracellular (SU) envelope glycoproteins with cellular receptors. To gain insight into the mechanisms governing this process, we investigated the ability of SU proteins to interact with specific ligands. In particular, by affinity chromatography, we have shown that BLV SU protein specifically interacted with zinc ions. To identify the protein domains involved in binding, 16 peptides distributed along the sequence were tested. Two of them appeared to be able to interact with zinc. To unravel the role of these SU regions in the biology of the virus, mutations were introduced into the env gene of a BLV molecular clone in order to modify residues potentially interacting with zinc. The fusogenic capacity of envelope mutated within the first zinc-binding region (104 to 123) was completely abolished. Furthermore, the integrity of this domain was also required for in vivo infectivity. In contrast, mutations within the second zinc-binding region (218 to 237) did not hamper the fusogenic capacity; indeed, the syncytia were even larger. In sheep, mutations in region 218 to 237 did not alter infectivity or viral spread. Finally, we demonstrated that the envelope of the related HTLV-1 was also able to bind zinc. Interestingly, zinc ions were found to be associated with the receptor-binding domain (RBD) of Friend murine leukemia virus (Fr-MLV) SU glycoprotein, further supporting their relevance in SU structure. Based on the sequence similarities shared with the Fr-MLV RBD, whose three-dimensional structure has been experimentally determined, we located the BLV zinc-binding peptide 104-123 on the opposite side of the potential receptor-binding surface. This observation supports the hypothesis that zinc ions could mediate interactions of the SU RBD either with the C-terminal part of SU, thereby contributing to the SU structural integrity, or with a partner(s) different from the receptor. PMID:12134000
-
von Stein, Richard T.; Soderlund, David M.
2012-01-01
Sodium channel inhibitor (SCI) insecticides are hypothesized to inhibit voltage-gated sodium channels by binding selectively to the slow-inactivated state. Replacement of valine at position 787 in the S6 segment of homology domain II of the rat Nav1.4 sodium channel by lysine (V787K) enchances slow inactivation of this channel whereas replacement by alanine or cysteine (V787A, V787C) inhibits slow inactivation. To test the hypothesis that SCI insecticides bind selectively to the slow-inactivated state, we constructed mutated Nav1.4/V787A, Nav1.4/V787C, and Nav1.4/V787K cDNAs, expressed wildtype and mutated channels with the auxiliary β1 subunit in Xenopus oocytes, and used the two-electrode voltage clamp technique to examine the effects of these mutations on channel inhibition by four SCI insecticides (indoxacarb, its bioactivated metabolite DCJW, metaflumizone, and RH3421). Mutations at Val787 affected SCI insecticide sensitivity in a manner that was independent of mutation-induced changes in slow inactivation gating. Sensitivity to inhibition by 10 μM indoxacarb was significantly increased in all three mutated channels, whereas sensitivity to inhibition by 10 μM metaflumizone was significantly reduced in Nav1.4/V787A channels and completely abolished in Nav1.4/V787K channels. The effects of Val787 mutations on metaflumizone were correlated with the hydrophobicity of the substituted amino acid rather than the extent of slow inactivation. None of the mutations at Val787 significantly affected the sensitivity to inhibition by DCJW or RH3421. These results demonstrate that the impact of mutations at Val787 on sodium channel inhibition by SCI insecticides depends on the specific insecticide examined and is independent of mutation-induced changes in slow inactivation gating. We propose that Val787 may be a unique determinant of metaflumizone binding. PMID:22983119
-
Effect of amino acid mutations on intra-dimer tubulin-tubulin binding strength of microtubules.
Liu, Ning; Pidaparti, Ramana; Wang, Xianqiao
2017-12-11
Energetic interactions inside αβ-tubulin dimers of a microtubule (MT) with atomic resolutions are of importance in determining the mechanical properties and structural stability of the MT as well as designing self-assembled functional structures from it. Here, we carry out several comprehensive atomistic simulations to investigate the interaction properties within αβ-tubulin dimers and effect of residue mutations on the intra-dimer tubulin-tubulin (IDTT) binding strength. Results indicate that the force-displacement responses of the dimer could be roughly divided into three stages involving increasing, decreasing, and fluctuating forces. Energetic analysis shows that electrostatic interactions dominate the IDTT binding strength. Further per-residue energetic analysis shows that the major part of the interface interaction energy (approximately 72% for α-tubulin and 62% for β-tubulin) comes from amino acid residues with net charges, namely arginine (ARG), lysine (LYS), glutamic acid (GLU), aspartic acid (ASP). Residue mutations are completed for ARG105 on α-tubulin and ASP251 on β-tubulin to study the effect of mutations on the IDTT binding strength. Results indicate that stiffness, rupture force, and interface interaction energy of αβ-tubulin dimer can be improved by up to 28%, 13% and 28%, respectively. Overall, our results provide a thorough atomistic understanding of the IDTT binding strength within αβ-tubulin heterodimers and help pave the way for eventually designing and controlling the self-assembled functional structures from MTs.
-
Zou, Changjiang; Levine, Edward M.
2012-01-01
The homeodomain and adjacent CVC domain in the visual system homeobox (VSX) proteins are conserved from nematodes to humans. Humans with missense mutations in these regions of VSX2 have microphthalmia, suggesting both regions are critical for function. To assess this, we generated the corresponding mutations in mouse Vsx2. The homeodomain mutant protein lacked DNA binding activity and the knock-in mutant phenocopied the null mutant, ocular retardation J. The CVC mutant protein exhibited weakened DNA binding; and, although the corresponding knock-in allele was recessive, it unexpectedly caused the strongest phenotype, as indicated by severe microphthalmia and hyperpigmentation of the neural retina. This occurred through a cryptic transcriptional feedback loop involving the transcription factors Mitf and Otx1 and the Cdk inhibitor p27Kip1. Our data suggest that the phenotypic severity of the CVC mutant depends on the weakened DNA binding activity elicited by the CVC mutation and a previously unknown protein interaction between Vsx2 and its regulatory target Mitf. Our data also suggest that an essential function of the CVC domain is to assist the homeodomain in high-affinity DNA binding, which is required for eye organogenesis and unhindered execution of the retinal progenitor program in mammals. Finally, the genetic and phenotypic behaviors of the CVC mutation suggest it has the characteristics of a recessive neomorph, a rare type of genetic allele. PMID:23028343
-
A molecular dynamics investigation into the mechanisms of alectinib resistance of three ALK mutants.
He, Muyang; Li, Weikang; Zheng, Qingchuan; Zhang, Hongxing
2018-07-01
Alectinib, a highly selective next-genetation anaplastic lymphoma kinase (ALK) inhibitor, has demonstrated promising antitumor activity in patients with ALK-positive non-small cell lung carcinomas (NSCLC). However, the therapeutic benefits of alectinib is inescapably hampered by the development of acquired resistant mutations in ALK. Despite the availability of ample experimental mutagenesis data, the molecular origin and the structural motifs under alectinib binding affinity deficiencies are still ambiguous. Here, molecular dynamics (MD) simulations and molecular mechanics generalized born surface area (MM-GBSA) calculation approaches were employed to elucidate the mechanisms of alectinib resistance induced by the mutations I1171N, V1180L, and L1198F. The MD results reveal that the studied mutations could trigger the dislocation of alectinib as well as conformational changes at the inhibitor binding site, thus induce the interactional changes between alectinib and mutants. The most influenced regions are the ligand binding entrance and the hinge region, which are considered to be the dominant binding motifs accounting for the binding affinity loss in mutants. The "key and lock mechanism" between the ethyl group at position 9 of alectinib and a recognition cavity in the hinge region of ALK is presented to illustrate the major molecular origin of drug resistance. Our results provide mechanistic insight into the effect of ALK mutations resistant to alectinib, which could contribute to further rational design of inhibitors to combat the acquired resistance. © 2018 Wiley Periodicals, Inc.
-
Montelone, Beth A.; Aguilera, Andrés
2014-01-01
The eukaryotic TFIIH complex is involved in Nucleotide Excision Repair and transcription initiation. We analyzed three yeast mutations of the Rad3/XPD helicase of TFIIH known as rem (recombination and mutation phenotypes). We found that, in these mutants, incomplete NER reactions lead to replication fork breaking and the subsequent engagement of the homologous recombination machinery to restore them. Nevertheless, the penetrance varies among mutants, giving rise to a phenotype gradient. Interestingly, the mutations analyzed reside at the ATP-binding groove of Rad3 and in vivo experiments reveal a gain of DNA affinity upon damage of the mutant Rad3 proteins. Since mutations at the ATP-binding groove of XPD in humans are present in the Xeroderma pigmentosum-Cockayne Syndrome (XP-CS), we recreated rem mutations in human cells, and found that these are XP-CS-like. We propose that the balance between the loss of helicase activity and the gain of DNA affinity controls the capacity of TFIIH to open DNA during NER, and its persistence at both DNA lesions and promoters. This conditions NER efficiency and transcription resumption after damage, which in human cells would explain the XP-CS phenotype, opening new perspectives to understand the molecular basis of the role of XPD in human disease. PMID:25500814
-
Mechanisms of FUS mutations in familial amyotrophic lateral sclerosis.
Shang, Yulei; Huang, Eric J
2016-09-15
Recent advances in the genetics of amyotrophic lateral sclerosis (ALS) have provided key mechanistic insights to the pathogenesis of this devastating neurodegenerative disease. Among many etiologies for ALS, the identification of mutations and proteinopathies in two RNA binding proteins, TDP-43 (TARDBP or TAR DNA binding protein 43) and its closely related RNA/DNA binding protein FUS (fused in sarcoma), raises the intriguing possibility that perturbations to the RNA homeostasis and metabolism in neurons may contribute to the pathogenesis of these diseases. Although the similarities between TDP-43 and FUS suggest that mutations and proteinopathy involving these two proteins may converge on the same mechanisms leading to neurodegeneration, there is increasing evidence that FUS mutations target distinct mechanisms to cause early disease onset and aggressive progression of disease. This review focuses on the recent advances on the molecular, cellular and genetic approaches to uncover the mechanisms of wild type and mutant FUS proteins during development and in neurodegeneration. These findings provide important insights to understand how FUS mutations may perturb the maintenance of dendrites through fundamental processes in RNA splicing, RNA transport and DNA damage response/repair. These results contribute to the understanding of phenotypic manifestations in neurodegeneration related to FUS mutations, and to identify important directions for future investigations. This article is part of a Special Issue entitled SI:RNA Metabolism in Disease. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.
-
Stanewsky, R.; Fry, T. A.; Reim, I.; Saumweber, H.; Hall, J. C.
1996-01-01
The no-on-transient-A (nonA) gene of Drosophila melanogaster influences vision, courtship song, and viability. The nonA-encoded polypeptide is inferred to bind single-stranded nucleic acids. Although sequence-analysis of NONA implies that it belongs to a special interspecific family of this protein type, it does contain two classical RNA recognition motifs (RRM). Their behavioral significance was assayed by generating transgenic strains that were singly or multiply mutated within the relatively N-terminal motif (RRM1) or within RRM2. Neither class of mutation affected NONA binding to polytene chromosomes. The former mutations led to extremely low viability, accompanied by diminished adult longevities that were much worse than for a nonA-null mutant, implying that faulty interpolypeptide interactions might accompany the effects of the amino-acid substitutions within RRM1. All in vitro-mutated types caused optomotor blindness and an absence of transient spikes in the electroretinogram. Courtship analysis discriminated between the effects of the mutations: the RRM2-mutated type generated song pulses and trains that tended to be mildly mutant. These phenotypic abnormalities reinforce the notion that nonA's ubiquitous expression has its most important consequences in the optic lobes, the thoracic ganglia, or both, depending in part on the nonA allele. PMID:8722780
-
Rezaei, Farideh; Shafiei, Mohammad; Shariati, Gholamreza; Dehdashtian, Ali; Mohebbi, Maryam; Galehdari, Hamid
2016-01-01
Introduction ABCA3 glycoprotein belongs to the ATP-binding cassette (ABC) superfamily of transporters, which utilize the energy derived from hydrolysis of ATP for the translocation of a wide variety of substrates across the plasma membrane. Mutations in the ABCA3 gene are knowingly causative for fatal surfactant deficiency, particularly respiratory distress syndrome (RDS) in term babies. Case Presentation In this study, Sanger sequencing of the whole ABCA3 gene (NCBI NM_001089) was performed in a neonatal boy with severe RDS. A homozygous mutation has been identified in the patient. Parents were heterozygous for the same missense mutation GGA > AGA at position 202 in exon 6 of the ABCA3 gene (c.604G > A; p.G202R). Furthermore, 70 normal individuals have been analyzed for the mentioned change with negative results. Conclusions Regarding Human Genome Mutation Database (HGMD) and other literature recherche, the detected change is a novel mutation and has not been reported before. Bioinformatics mutation predicting tools prefer it as pathogenic. PMID:27437095
-
Rett Syndrome Mutation MeCP2 T158A Disrupts DNA Binding, Protein Stability and ERP Responses
Goffin, Darren; Allen, Megan; Zhang, Le; Amorim, Maria; Wang, I-Ting Judy; Reyes, Arith-Ruth S.; Mercado-Berton, Amy; Ong, Caroline; Cohen, Sonia; Hu, Linda; Blendy, Julie A.; Carlson, Gregory C.; Siegel, Steve J.; Greenberg, Michael E.; Zhou, Zhaolan (Joe)
2011-01-01
Mutations in the MECP2 gene cause the autism spectrum disorder Rett Syndrome (RTT). One of the most common mutations associated with RTT occurs at MeCP2 Threonine 158 converting it to Methionine (T158M) or Alanine (T158A). To understand the role of T158 mutation in the pathogenesis of RTT, we generated knockin mice recapitulating MeCP2 T158A mutation. Here we show a causal role for T158A mutation in the development of RTT-like phenotypes including developmental regression, motor dysfunction, and learning and memory deficits. These phenotypes resemble those in Mecp2-null mice and manifest through a reduction in MeCP2 binding to methylated DNA and a decrease in MeCP2 protein stability. Importantly, the age-dependent development of event-related neuronal responses are disrupted by MeCP2 mutation, suggesting that impaired neuronal circuitry underlies the pathogenesis of RTT and that assessment of event-related potentials may serve as a biomarker for RTT and treatment evaluation. PMID:22119903
-
DOE Office of Scientific and Technical Information (OSTI.GOV)
Griffith, A.J.; Burgess, D.L.; Kohrman, D.
1994-09-01
The Twirler mutation (Tw) causing cleft palate {plus_minus} cleft lip, vestibular defects and obesity is located within 0.5 cM of an ataxia locus (ax) on mouse chromosome 18. We identified a transgene-induced insertional mutation with vestibular and craniofacial defects that appears to be a new allele of Twirler. Mouse DNA flanking the transgene insertion site was isolated from a cosmid library. An evolutionarily conserved, zoo blot positive cosmid subclone was used to probe a human {lambda} genomic library. From the sequence of a highly homologous human {lambda} clone, we designed STS primers and screened a human P1 library. DNA frommore » two positive P1 clones was hybridized with simple sequence probes, and a (CTAT){sub 12} repeat was detected. Analysis of 62 CEPH parents with primers flanking the repeat identified six alleles containing 9 to 14 copies of the repeat, at frequencies of 0.17, 0.17, 0.17, 0.27, 0.15 and 0.07, respectively. The observed heterozygosity was 49/62 with a calculated PIC value of 0.76. This polymorphic microsatellite marker, designated Umi3, was mapped to the predicted conserved human linkage group by analysis of somatic cell hybrid panels. The anticipated short distance between Umi3 and the disease genes will facilitate detection of linkage in small families. We would like to type appropriate human pedigrees with Umi3 in order to identify patients with inherited disorders homologous to the mouse mutations Twirler and ataxia.« less
-
Functional map of arrestin binding to phosphorylated opsin, with and without agonist.
Peterhans, Christian; Lally, Ciara C M; Ostermaier, Martin K; Sommer, Martha E; Standfuss, Jörg
2016-06-28
Arrestins desensitize G protein-coupled receptors (GPCRs) and act as mediators of signalling. Here we investigated the interactions of arrestin-1 with two functionally distinct forms of the dim-light photoreceptor rhodopsin. Using unbiased scanning mutagenesis we probed the individual contribution of each arrestin residue to the interaction with the phosphorylated apo-receptor (Ops-P) and the agonist-bound form (Meta II-P). Disruption of the polar core or displacement of the C-tail strengthened binding to both receptor forms. In contrast, mutations of phosphate-binding residues (phosphosensors) suggest the phosphorylated receptor C-terminus binds arrestin differently for Meta II-P and Ops-P. Likewise, mutations within the inter-domain interface, variations in the receptor-binding loops and the C-edge of arrestin reveal different binding modes. In summary, our results indicate that arrestin-1 binding to Meta II-P and Ops-P is similarly dependent on arrestin activation, although the complexes formed with these two receptor forms are structurally distinct.
-
Genome-Wide Motif Statistics are Shaped by DNA Binding Proteins over Evolutionary Time Scales
NASA Astrophysics Data System (ADS)
Qian, Long; Kussell, Edo
The composition of genomes with respect to short DNA motifs impacts the ability of DNA binding proteins to locate and bind their target sites. Since nonfunctional DNA binding can be detrimental to cellular functions and ultimately to organismal fitness, organisms could benefit from reducing the number of nonfunctional binding sites genome wide. Using in vitro measurements of binding affinities for a large collection of DNA binding proteins, in multiple species, we detect a significant global avoidance of weak binding sites in genomes. The underlying evolutionary process leaves a distinct genomic hallmark in that similar words have correlated frequencies, which we detect in all species across domains of life. We hypothesize that natural selection against weak binding sites contributes to this process, and using an evolutionary model we show that the strength of selection needed to maintain global word compositions is on the order of point mutation rates. Alternative contributions may come from interference of protein-DNA binding with replication and mutational repair processes, which operates with similar rates. We conclude that genome-wide word compositions have been molded by DNA binding proteins through tiny evolutionary steps over timescales spanning millions of generations.
-
NASA Astrophysics Data System (ADS)
Ishioka, T.; Yamada, H.; Miyakawa, T.; Morikawa, R.; Akanuma, S.; Yamagishi, A.; Takasu, M.
2016-12-01
Proteins, which incorporate charged and hydrophobic amino acid residues, are useful as a material of nanotechnology. Among these proteins, IPMDH (3-isopropylmalate dehydrogenase), which has thermal stability, has potential as a material of nanofiber. In this study, we performed coarse-grained molecular dynamics simulation of IPMDH using MARTINI force fields, and we investigated the orientation for the binding of IPMDH. In simulation, we analyzed wild type of IPMDH and the mutated IPMDH proteins, where 13, 20, 27, 332, 335 and 338th amino acid residues are replaced by lysine residues which have positive charge and by glutamic acid residues which have negative charge. Since the binding of mutated IPMDH is advantageous compared with the binding of wild type for one orientation, we suggest that the Coulomb interaction for the binding of IPMDH is important.
-
Biochemical analysis of human POLG2 variants associated with mitochondrial disease
Young, Matthew J.; Longley, Matthew J.; Li, Fang-Yuan; Kasiviswanathan, Rajesh; Wong, Lee-Jun; Copeland, William C.
2011-01-01
Defects in mitochondrial DNA (mtDNA) maintenance comprise an expanding repertoire of polymorphic diseases caused, in part, by mutations in the genes encoding the p140 mtDNA polymerase (POLG), its p55 accessory subunit (POLG2) or the mtDNA helicase (C10orf2). In an exploration of nuclear genes for mtDNA maintenance linked to mitochondrial disease, eight heterozygous mutations (six novel) in POLG2 were identified in one control and eight patients with POLG-related mitochondrial disease that lacked POLG mutations. Of these eight mutations, we biochemically characterized seven variants [c.307G>A (G103S); c.457C>G (L153V); c.614C>G (P205R); c.1105A>G (R369G); c.1158T>G (D386E); c.1268C>A (S423Y); c.1423_1424delTT (L475DfsX2)] that were previously uncharacterized along with the wild-type protein and the G451E pathogenic variant. These seven mutations encode amino acid substitutions that map throughout the protein, including the p55 dimer interface and the C-terminal domain that interacts with the catalytic subunit. Recombinant proteins harboring these alterations were assessed for stimulation of processive DNA synthesis, binding to the p140 catalytic subunit, binding to dsDNA and self-dimerization. Whereas the G103S, L153V, D386E and S423Y proteins displayed wild-type behavior, the P205R and R369G p55 variants had reduced stimulation of processivity and decreased affinity for the catalytic subunit. Additionally, the L475DfsX2 variant, which possesses a C-terminal truncation, was unable to bind the p140 catalytic subunit, unable to bind dsDNA and formed aberrant oligomeric complexes. Our biochemical analysis helps explain the pathogenesis of POLG2 mutations in mitochondrial disease and emphasizes the need to quantitatively characterize the biochemical consequences of newly discovered mutations before classifying them as pathogenic. PMID:21555342
-
Ali, Bassam R; Xu, Huifang; Akawi, Nadia A; John, Anne; Karuvantevida, Noushad S; Langer, Ruth; Al-Gazali, Lihadh; Leitinger, Birgit
2010-06-01
Spondylo-meta-epiphyseal dysplasia (SMED) with short limbs and abnormal calcifications (SMED-SL) is a rare, autosomal recessive human growth disorder, characterized by disproportionate short stature, short limbs, short broad fingers, abnormal metaphyses and epiphyses, platyspondyly and premature calcifications. Recently, three missense mutations and one splice-site mutation in the DDR2 gene were identified as causative genetic defects for SMED-SL, but the underlying cellular and biochemical mechanisms were not explored. Here we report a novel DDR2 missense mutation, c.337G>A (p.E113K), that causes SMED-SL in two siblings in the United Arab Emirates. Another DDR2 missense mutation, c.2254C>T (p.R752C), matching one of the previously reported SMED-SL mutations, was found in a second affected family. DDR2 is a plasma membrane receptor tyrosine kinase that functions as a collagen receptor. We expressed DDR2 constructs with the identified point mutations in human cell lines and evaluated their localization and functional properties. We found that all SMED-SL missense mutants were defective in collagen-induced receptor activation and that the three previously reported mutants (p.T713I, p.I726R and p.R752C) were retained in the endoplasmic reticulum. The novel mutant (p.E113K), in contrast, trafficked normally, like wild-type DDR2, but failed to bind collagen. This finding is in agreement with our recent structural data identifying Glu113 as an important amino acid in the DDR2 ligand-binding site. Our data thus demonstrate that SMED-SL can result from at least two different loss-of-function mechanisms: namely defects in DDR2 targeting to the plasma membrane or the loss of its ligand-binding activity.
-
Ali, Bassam R.; Xu, Huifang; Akawi, Nadia A.; John, Anne; Karuvantevida, Noushad S.; Langer, Ruth; Al-Gazali, Lihadh; Leitinger, Birgit
2010-01-01
Spondylo-meta-epiphyseal dysplasia (SMED) with short limbs and abnormal calcifications (SMED-SL) is a rare, autosomal recessive human growth disorder, characterized by disproportionate short stature, short limbs, short broad fingers, abnormal metaphyses and epiphyses, platyspondyly and premature calcifications. Recently, three missense mutations and one splice-site mutation in the DDR2 gene were identified as causative genetic defects for SMED-SL, but the underlying cellular and biochemical mechanisms were not explored. Here we report a novel DDR2 missense mutation, c.337G>A (p.E113K), that causes SMED-SL in two siblings in the United Arab Emirates. Another DDR2 missense mutation, c.2254C>T (p.R752C), matching one of the previously reported SMED-SL mutations, was found in a second affected family. DDR2 is a plasma membrane receptor tyrosine kinase that functions as a collagen receptor. We expressed DDR2 constructs with the identified point mutations in human cell lines and evaluated their localization and functional properties. We found that all SMED-SL missense mutants were defective in collagen-induced receptor activation and that the three previously reported mutants (p.T713I, p.I726R and p.R752C) were retained in the endoplasmic reticulum. The novel mutant (p.E113K), in contrast, trafficked normally, like wild-type DDR2, but failed to bind collagen. This finding is in agreement with our recent structural data identifying Glu113 as an important amino acid in the DDR2 ligand-binding site. Our data thus demonstrate that SMED-SL can result from at least two different loss-of-function mechanisms: namely defects in DDR2 targeting to the plasma membrane or the loss of its ligand-binding activity. PMID:20223752
-
2011-01-01
Background Well differentiated papillary mesothelioma of the peritoneum (WDPMP) is a rare variant of epithelial mesothelioma of low malignancy potential, usually found in women with no history of asbestos exposure. In this study, we perform the first exome sequencing of WDPMP. Results WDPMP exome sequencing reveals the first somatic mutation of E2F1, R166H, to be identified in human cancer. The location is in the evolutionarily conserved DNA binding domain and computationally predicted to be mutated in the critical contact point between E2F1 and its DNA target. We show that the R166H mutation abrogates E2F1's DNA binding ability and is associated with reduced activation of E2F1 downstream target genes. Mutant E2F1 proteins are also observed in higher quantities when compared with wild-type E2F1 protein levels and the mutant protein's resistance to degradation was found to be the cause of its accumulation within mutant over-expressing cells. Cells over-expressing wild-type E2F1 show decreased proliferation compared to mutant over-expressing cells, but cell proliferation rates of mutant over-expressing cells were comparable to cells over-expressing the empty vector. Conclusions The R166H mutation in E2F1 is shown to have a deleterious effect on its DNA binding ability as well as increasing its stability and subsequent accumulation in R166H mutant cells. Based on the results, two compatible theories can be formed: R166H mutation appears to allow for protein over-expression while minimizing the apoptotic consequence and the R166H mutation may behave similarly to SV40 large T antigen, inhibiting tumor suppressive functions of retinoblastoma protein 1. PMID:21955916
-
Namour, Fares; Dobrovoljski, Gabriele; Chery, Celine; Audonnet, Sandra; Feillet, François; Sperl, Wolfgang; Gueant, Jean-Louis
2011-01-01
Juvenile megaloblastic anaemia 1 (OMIM # 261100) is a rare autosomic disorder characterized by selective cobalamin mal-absorption and inconstant proteinuria produced by mutations in either CUBN or AMN genes. Amnionless, the gene product of AMN, is a transmembrane protein that binds tightly to the N-terminal end of cubilin, the gene product of CUBN. Cubilin binds to intrinsic factor-cobalamin complex and is expressed in the distal intestine and the proximal renal tubule. We report a compound AMN heterozygosity with c.742C>T, p.Gln248X and c.208-2A>G mutations in 2 siblings that led to premature termination codon in exon 7 and exon 6, respectively. It produced a dramatic decrease in receptor activity in urine, despite absence of CUBN mutation and normal affinity of the receptor for intrinsic factor binding. Heterozygous carriers for c.742T and c.208-2G had no pathological signs. These results indicate that amnionless is essential for the correct luminal expression of cubilin in humans. PMID:21750092
-
Structural Determinants for Naturally Evolving H5N1 Hemagglutinin to Switch its Receptor Specificity
Tharakaraman, Kannan; Raman, Rahul; Viswanathan, Karthik; Stebbins, Nathan W.; Jayaraman, Akila; Krishnan, Arvind; Sasisekharan, V.; Sasisekharan, Ram
2013-01-01
SUMMARY Of the factors governing human-to-human transmission of the highly pathogenic avian-adapted H5N1 virus, the most critical is the acquisition of mutations on the viral hemagglutinin (HA) to “quantitatively switch” its binding from avian to human glycan receptors. Herein, we describe a structural framework that outlines a necessary set of H5 HA receptor binding site (RBS) features required for the H5 HA to quantitatively switch its preference to human receptors. We show here that the same RBS HA mutations that lead to aerosol transmission of A/Vietnam/1203/04 and A/Indonesia/5/05 viruses, when introduced in currently circulating H5N1, do not lead to quantitative switch in receptor preference. We demonstrate that HAs from circulating clades require as few as a single base-pair mutation to quantitatively switch their binding to human receptors. The mutations identified by this study can be used to monitor the emergence of strains having human-to-human transmission potential. PMID:23746829
-
Mild and severe muscular dystrophy caused by a single {gamma}-sarcoglycan mutation
DOE Office of Scientific and Technical Information (OSTI.GOV)
McNally, E.M.; Boennemann, C.G.; Lidov, H.G.W.
1996-11-01
Autosomal recessive muscular dystrophy is genetically heterogeneous. One form of this disorder, limb-girdle muscular dystrophy type 2C (LGMD 2C), is prevalent in northern Africa and has been shown to be associated with a single mutation in the gene encoding the dystrophin-associated protein {gamma}-sarcoglycan. The previous mutation analysis of {gamma}-sarcoglycan required the availability of muscle biopsies. To establish a mutation assay for genomic DNA, the intron-exon structure of the {gamma}-sarcoglycan gene was determined, and primers were designed to amplify each of the exons encoding {gamma}-sarcoglycan. We studied a group of Brazilian muscular dystrophy patients for mutations in the {gamma}-sarcoglycan gene. Thesemore » patients were selected on the basis of autosomal inheritance and/or the presence of normal dystrophin and/or deficiency of {alpha}-sarcoglycan immunostaining. Four of 19 patients surveyed had a single, homozygous mutation in the {gamma}-sarcoglycan gene. The mutation identified in these patients, all of African-Brazilian descent, is identical to that seen in the North African population, suggesting that even patients of remote African descent may carry this mutation. The phenotype in these patients varied considerably. Of four families with an identical mutation, three have a severe Duchenne-like muscular dystrophy. However, one family has much milder symptoms, suggesting that other loci may be present that modify the severity of the clinical course resulting from {gamma}-sarcoglycan gene mutations. 19 refs., 5 figs., 3 tabs.« less
-
Usher syndrome in Denmark: mutation spectrum and some clinical observations.
Dad, Shzeena; Rendtorff, Nanna Dahl; Tranebjærg, Lisbeth; Grønskov, Karen; Karstensen, Helena Gásdal; Brox, Vigdis; Nilssen, Øivind; Roux, Anne-Françoise; Rosenberg, Thomas; Jensen, Hanne; Møller, Lisbeth Birk
2016-09-01
Usher syndrome (USH) is a genetically heterogeneous deafness-blindness syndrome, divided into three clinical subtypes: USH1, USH2 and USH3. Mutations in 21 out of 26 investigated Danish unrelated individuals with USH were identified, using a combination of molecular diagnostic methods. Before Next Generation Sequencing (NGS) became available mutations in nine individuals (1 USH1, 7 USH2, 1 USH3) were identified by Sanger sequencing of USH1C , USH2A or CLRN1 or by Arrayed Primer EXtension (APEX) method. Mutations in 12 individuals (7 USH1, 5 USH2) were found by targeted NGS of ten known USH genes. Five novel pathogenic variants were identified. We combined our data with previously published, and obtained an overview of the USH mutation spectrum in Denmark, including 100 unrelated individuals; 32 with USH1, 67 with USH2, and 1 with USH3. Macular edema was observed in 44 of 117 individuals. Olfactory function was tested in 12 individuals and found to be within normal range in all. Mutations that lead to USH1 were predominantly identified in MYO7A (75%), whereas all mutations in USH2 cases were identified in USH2A . The MYO7A mutation c.93C>A, p.(Cys31*) accounted for 33% of all USH1 mutations and the USH2A c.2299delG, p.(Glu767Serfs*21) variant accounted for 45% of all USH2 mutations in the Danish cohort.
-
Mutations in FLNB cause boomerang dysplasia
Bicknell, L; Morgan, T; Bonafe, L; Wessels, M; Bialer, M; Willems, P; Cohn, D; Krakow, D; Robertson, S
2005-01-01
Boomerang dysplasia (BD) is a perinatal lethal osteochondrodysplasia, characterised by absence or underossification of the limb bones and vertebrae. The BD phenotype is similar to a group of disorders including atelosteogenesis I, atelosteogenesis III, and dominantly inherited Larsen syndrome that we have recently shown to be associated with mutations in FLNB, the gene encoding the actin binding cytoskeletal protein, filamin B. We report the identification of mutations in FLNB in two unrelated individuals with boomerang dysplasia. The resultant substitutions, L171R and S235P, lie within the calponin homology 2 region of the actin binding domain of filamin B and occur at sites that are evolutionarily well conserved. These findings expand the phenotypic spectrum resulting from mutations in FLNB and underline the central role this protein plays during skeletogenesis in humans. PMID:15994868
-
Mutations in FLNB cause boomerang dysplasia.
Bicknell, L S; Morgan, T; Bonafé, L; Wessels, M W; Bialer, M G; Willems, P J; Cohn, D H; Krakow, D; Robertson, S P
2005-07-01
Boomerang dysplasia (BD) is a perinatal lethal osteochondrodysplasia, characterised by absence or underossification of the limb bones and vertebrae. The BD phenotype is similar to a group of disorders including atelosteogenesis I, atelosteogenesis III, and dominantly inherited Larsen syndrome that we have recently shown to be associated with mutations in FLNB, the gene encoding the actin binding cytoskeletal protein, filamin B. We report the identification of mutations in FLNB in two unrelated individuals with boomerang dysplasia. The resultant substitutions, L171R and S235P, lie within the calponin homology 2 region of the actin binding domain of filamin B and occur at sites that are evolutionarily well conserved. These findings expand the phenotypic spectrum resulting from mutations in FLNB and underline the central role this protein plays during skeletogenesis in humans.
-
Xue, Weiwei; Jiao, Pingzu; Liu, Huanxiang; Yao, Xiaojun
2014-04-01
Hepatitis C virus (HCV) NS5B protein is an RNA-dependent RNA polymerase (RdRp) with essential functions in viral genome replication and represents a promising therapeutic target to develop direct-acting antivirals (DAAs). Multiple nonnucleoside inhibitors (NNIs) binding sites have been identified within the polymerase. VX-222 and ANA598 are two NNIs targeting thumb II site and palm I site of HCV NS5B polymerase, respectively. These two molecules have been shown to be very effective in phase II clinical trials. However, the emergence of resistant HCV replicon variants (L419M, M423T, I482L mutants to VX-222 and M414T, M414L, G554D mutants to ANA598) has significantly decreased their efficacy. To elucidate the molecular mechanism about how these mutations influenced the drug binding mode and decreased drug efficacy, we studied the binding modes of VX-222 and ANA598 to wild-type and mutant polymerase by molecular modeling approach. Molecular dynamics (MD) simulations results combined with binding free energy calculations indicated that the mutations significantly altered the binding free energy and the interaction for the drugs to polymerase. The further per-residue binding free energy decomposition analysis revealed that the mutations decreased the interactions with several key residues, such as L419, M423, L474, S476, I482, L497, for VX-222 and L384, N411, M414, Y415, Q446, S556, G557 for ANA598. These were the major origins for the resistance to these two drugs. In addition, by analyzing the residue interaction network (RIN) of the complexes between the drugs with wild-type and the mutant polymerase, we found that the mutation residues in the networks involved in the drug resistance possessed a relatively lower size of topology centralities. The shift of betweenness and closeness values of binding site residues in the mutant polymerase is relevant to the mechanism of drug resistance of VX-222 and ANA598. These results can provide an atomic-level understanding about the mechanisms of drug resistance conferred by the studied mutations and will be helpful to design more potent inhibitors which could effectively overcome drug resistance of antivirus agents. Copyright © 2014 Elsevier B.V. All rights reserved.
-
Li, Xianting; Wang, Qing Jun; Pan, Nina; Lee, Sangkyu; Zhao, Yingming; Chait, Brian T.; Yue, Zhenyu
2011-01-01
Background Recent studies show that mutations in Leucine Rich Repeat Kinase 2 (LRRK2) are the cause of the most common inherited and some sporadic forms of Parkinson's disease (PD). The molecular mechanism underlying the pathogenic role of LRRK2 mutations in PD remains unknown. Methodology/Principal Findings Using affinity purification and mass spectrometric analysis, we investigated phosphorylation sites and binding proteins of LRRK2 purified from mouse brain. We identified multiple phosphorylation sites at N-terminus of LRRK2 including S910, S912, S935 and S973. Focusing on the high stoichiometry S935 phosphorylation site, we developed an anti-pS935 specific antibody and showed that LRRK2 is constitutively phosphorylated at S935 in various tissues (including brain) and at different ages in mice. We find that 14-3-3 proteins (especially isoforms γ and η) bind LRRK2 and this binding depends on phosphorylation of S935. The binding of 14-3-3, with little effect on dimer formation of LRRK2, confers protection of the phosphorylation status of S935. Furthermore, we show that protein kinase A (PKA), but not LRRK2 kinase itself, can cause the phosphorylation of LRRK2 at S935 in vitro and in cell culture, suggesting that PKA is a potential upstream kinase that regulates LRRK2 function. Finally, our study indicates that the common PD-related mutations of LRRK2, R1441G, Y1699C and G2019S, decrease homeostatic phosphorylation levels of S935 and impair 14-3-3 binding of LRRK2. Conclusions/Significance LRRK2 is extensively phosphorylated in vivo, and the phosphorylation of specific sites (e.g. S935) determines 14-3-3 binding of LRRK2. We propose that 14-3-3 is an important regulator of LRRK2-mediated cellular functions. Our study suggests that PKA, a cAMP-dependent kinase involved in regulating dopamine physiology, is a potential upstream kinase that phosphorylates LRRK2 at S935. Furthermore, the reduction of phosphorylation/14-3-3 binding of LRRK2 due to the common familial PD-related mutations provides novel insight into the pathogenic mechanism of LRRK2-linked PD. PMID:21390248
-
Gonadotropin-Releasing Hormone (GnRH) Receptor Structure and GnRH Binding
Flanagan, Colleen A.; Manilall, Ashmeetha
2017-01-01
Gonadotropin-releasing hormone (GnRH) regulates reproduction. The human GnRH receptor lacks a cytoplasmic carboxy-terminal tail but has amino acid sequence motifs characteristic of rhodopsin-like, class A, G protein-coupled receptors (GPCRs). This review will consider how recent descriptions of X-ray crystallographic structures of GPCRs in inactive and active conformations may contribute to understanding GnRH receptor structure, mechanism of activation and ligand binding. The structures confirmed that ligands bind to variable extracellular surfaces, whereas the seven membrane-spanning α-helices convey the activation signal to the cytoplasmic receptor surface, which binds and activates heterotrimeric G proteins. Forty non-covalent interactions that bridge topologically equivalent residues in different transmembrane (TM) helices are conserved in class A GPCR structures, regardless of activation state. Conformation-independent interhelical contacts account for a conserved receptor protein structure and their importance in the GnRH receptor structure is supported by decreased expression of receptors with mutations of residues in the network. Many of the GnRH receptor mutations associated with congenital hypogonadotropic hypogonadism, including the Glu2.53(90) Lys mutation, involve amino acids that constitute the conserved network. Half of the ~250 intramolecular interactions in GPCRs differ between inactive and active structures. Conformation-specific interhelical contacts depend on amino acids changing partners during activation. Conserved inactive conformation-specific contacts prevent receptor activation by stabilizing proximity of TM helices 3 and 6 and a closed G protein-binding site. Mutations of GnRH receptor residues involved in these interactions, such as Arg3.50(139) of the DRY/S motif or Tyr7.53(323) of the N/DPxxY motif, increase or decrease receptor expression and efficiency of receptor coupling to G protein signaling, consistent with the native residues stabilizing the inactive GnRH receptor structure. Active conformation-specific interhelical contacts stabilize an open G protein-binding site. Progress in defining the GnRH-binding site has recently slowed, with evidence that Tyr6.58(290) contacts Tyr5 of GnRH, whereas other residues affect recognition of Trp3 and Gly10NH2. The surprisingly consistent observations that GnRH receptor mutations that disrupt GnRH binding have less effect on “conformationally constrained” GnRH peptides may now be explained by crystal structures of agonist-bound peptide receptors. Analysis of GPCR structures provides insight into GnRH receptor function. PMID:29123501
-
Transposon facilitated DNA sequencing
DOE Office of Scientific and Technical Information (OSTI.GOV)
Berg, D.E.; Berg, C.M.; Huang, H.V.
1990-01-01
The purpose of this research is to investigate and develop methods that exploit the power of bacterial transposable elements for large scale DNA sequencing: Our premise is that the use of transposons to put primer binding sites randomly in target DNAs should provide access to all portions of large DNA fragments, without the inefficiencies of methods involving random subcloning and attendant repetitive sequencing, or of sequential synthesis of many oligonucleotide primers that are used to match systematically along a DNA molecule. Two unrelated bacterial transposons, Tn5 and {gamma}{delta}, are being used because they have both proven useful for molecular analyses,more » and because they differ sufficiently in mechanism and specificity of transposition to merit parallel development.« less
-
Ramey, Jordan D.; Villareal, Valerie A.; Ng, Charles; Ward, Sabrina; Xiong, Jian-Ping; Clubb, Robert T.; Bradley, Kenneth A.
2010-01-01
Anthrax toxin receptor 1 (ANTXR1) / tumor endothelial marker 8 (TEM8) is one of two known proteinaceous cell surface anthrax toxin receptors. A metal ion dependent adhesion site (MIDAS) present in the integrin-like inserted (I) domain of ANTXR1 mediates the binding of the anthrax toxin subunit, protective antigen (PA). Here we provide evidence that single point mutations in the I domain can override regulation of ANTXR1 ligand-binding activity mediated by intracellular signals. A previously reported MIDAS-mutant of ANTXR1 (T118A) was found to retain normal metal ion binding and secondary structure but failed to bind PA, consistent with a locked inactive state. Conversely, mutation of a conserved I domain phenylalanine residue to a tryptophan (F205W) increased the proportion of cell-surface ANTXR1 that bound PA, consistent with a locked active state. Interestingly, the KD and total amount of PA bound by the isolated ANTXR1 I domain was not affected by the F205W mutation, indicating that ANTXR1 is preferentially found in the active state in the absence of inside-out signaling. Circular dichroism (CD) spectroscopy and 1H-15N heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) revealed that structural changes between T118A, F205W and WT I domains were minor despite a greater than 103-fold difference in their abilities to bind toxin. Regulation of toxin binding has important implications for the design of toxin inhibitors and for the targeting of ANTXR1 for anti-tumor therapies. PMID:20690680