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Ozdemir, D., P. S. Hart, O. H. Ryu, S. J. Choi, M. Ozdemir-Karatas, E. Firatli, N. Piesco i T. C. Hart. "MMP20 Active-site Mutation in Hypomaturation Amelogenesis Imperfecta". Journal of Dental Research 84, nr 11 (listopad 2005): 1031–35. http://dx.doi.org/10.1177/154405910508401112.
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The Amelogenesis Imperfecta (AI) are a group of clinically and genetically heterogeneous disorders that affect enamel formation. To date, mutations in 4 genes have been reported in various types of AI. Mutations in the genes encoding the 2 enamel proteases, matrix metalloproteinase 20 ( MMP20) and kallikrein 4 ( KLK4), have each been reported in a single family segregating autosomal-recessive hypomaturation AI. To determine the frequency of mutations in these genes, we analyzed 15 Turkish probands with autosomal-recessive hypomaturation AI for MMP20 and KLK4 gene mutations. No KLK4 mutations were found. A novel MMP20 mutation (g.16250T>A) was found in one family. This missense mutation changed the conserved active-site His226 residue of the zinc catalytic domain to Gln (p.H226Q). Zymogram analysis demonstrated that this missense mutation abolished MMP20 proteolytic activity. No MMP20 mutations were found in the remaining 14 probands, underscoring the genetic heterogeneity of hypomaturation AI.
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Bianchi, F., S. Rosati, L. Belvederesi, C. Loretelli, R. Catalani, A. Mandolesi, R. Bracci, I. Bearzi, E. Porfiri i R. Cellerino. "MSH2 splice site mutation and endometrial cancer". International Journal of Gynecologic Cancer 16, nr 3 (2006): 1419–23. http://dx.doi.org/10.1136/ijgc-00009577-200605000-00072.
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Hereditary nonpolyposis colorectal cancer (HNPCC) is an inherited syndrome of cancer susceptibility caused by germ line mutations of genes participating in mismatch repair (MMR). Carriers of MMR gene mutations have an increased risk of colorectal cancers and cancer of other organs. Tumors of the endometrium represent the most frequent extracolonic malignancies in HNPCC. It has been suggested that women harboring MMR gene mutations have a higher risk of endometrial cancer than of colon cancer. Here, we describe an HNPCC patient with early-onset endometrial cancer and a strong familial history of endometrial tumors who harbored a germ line MSH2 splice site mutation (IVS9_2A>G). This mutation was responsible for abnormal messenger RNA processing, leading to the introduction of a premature stop signal and to the expression of a truncated MSH2 protein. In addition, the same mutation was associated with loss of MSH2 protein expression, high microsatellite instability, and PTEN inactivation. Although a direct relationship between the endometrial cancer susceptibility and the MSH2 mutation we found cannot be established, our observations, consistent with the work of other authors, suggest the involvement of germ line MSH2 abnormalities in endometrial tumor development and support the case for endometrial cancer screening in women from HNPCC families.
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Agosto, Melina A., Jason K. Middleton, Elaine C. Freimont, John Yin i Max L. Nibert. "Thermolabilizing Pseudoreversions in Reovirus Outer-Capsid Protein μ1 Rescue the Entry Defect Conferred by a Thermostabilizing Mutation". Journal of Virology 81, nr 14 (16.05.2007): 7400–7409. http://dx.doi.org/10.1128/jvi.02720-06.
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ABSTRACT Heat-resistant mutants selected from infectious subvirion particles of mammalian reoviruses have determinative mutations in the major outer-capsid protein μ1. Here we report the isolation and characterization of intragenic pseudoreversions of one such thermostabilizing mutation. From a plaque that had survived heat selection, a number of viruses with one shared mutation but different second-site mutations were isolated. The effect of the shared mutation alone or in combination with second-site mutations was examined using recoating genetics. The shared mutation, D371A, was found to confer (i) substantial thermostability, (ii) an infectivity defect that followed attachment but preceded viral protein synthesis, and (iii) resistance to μ1 rearrangement in vitro, with an associated failure to lyse red blood cells. Three different second-site mutations were individually tested in combination with D371A and found to wholly or partially revert these phenotypes. Furthermore, when tested alone in recoated particles, each of these three second-site mutations conferred demonstrable thermolability. This and other evidence suggest that pseudoreversion of μ1-based thermostabilization can occur by a general mechanism of μ1-based thermolabilization, not requiring a specific compensatory mutation. The thermostabilizing mutation D371A as well as 9 of the 10 identified second-site mutations are located near contact regions between μ1 trimers in the reovirus outer capsid. The availability of both thermostabilizing and thermolabilizing mutations in μ1 should aid in defining the conformational rearrangements and mechanisms involved in membrane penetration during cell entry by this structurally complex nonenveloped animal virus.
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Bauer, C. E., J. F. Gardner, R. I. Gumport i R. A. Weisberg. "The effect of attachment site mutations on strand exchange in bacteriophage lambda site-specific recombination." Genetics 122, nr 4 (1.08.1989): 727–36. http://dx.doi.org/10.1093/genetics/122.4.727.
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Abstract Recombination of phage lambda attachment sites occurs by sequential exchange of the DNA strands at two specific locations. The first exchange produces a Holliday structure, and the second resolves it to recombinant products. Heterology for base substitution mutations in the region between the two strand exchange points (the overlap region) reduces recombination; some mutations inhibit the accumulation of Holliday structures, others inhibit their resolution to recombinant products. To see if heterology also alters the location of the strand exchange points, we determined the segregation pattern of three single and one multiple base pair substitution mutations of the overlap region in crosses with wild type sites. The mutations are known to differ in the severity of their recombination defect and in the stage of strand exchange they affect. The three single mutations behaved similarly: each segregated into both products of recombination, and the two products of a single crossover were frequently nonreciprocal in the overlap region. In contrast, the multiple mutation preferentially segregated into one of the two recombinant products, and the two products of a single crossover appeared to be fully reciprocal. The simplest explanation of the segregation pattern of the single mutations is that strand exchanges occur at the normal locations to produce recombinants with mismatched base pairs that are frequently repaired. The segregation pattern of the multiple mutation is consistent with the view that both strand exchanges usually occur to one side of the mutant site. We suggest that the segregation pattern of a particular mutation is determined by which stage of strand exchange it inhibits and by the severity of the inhibition.
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Joseph, Ranjit, Paul Little, David N. Hayes i Michael Sangmin Lee. "Characterization of the number and site of APC mutations in sporadic colorectal cancer." Journal of Clinical Oncology 35, nr 4_suppl (1.02.2017): 630. http://dx.doi.org/10.1200/jco.2017.35.4_suppl.630.
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630 Background: Truncating mutations in the adenomatous polyposis coli ( APC) gene are well-described events in the carcinogenesis of colorectal carcinomas (CRC) and may impact one or both APC alleles. These aberrations often fall within the mutation cluster region (MCR) of the APC gene to preserve a “just right” number of beta-catenin binding sites in the resulting mutant APC protein. Further clinical and genotypic characterization of CRCs based on number and site of mutations in APC determined using modern next generation sequencing methods is needed. Methods: Next generation sequencing of 70 CRC tumors was performed at a single institution via UNCseq to determine mutations in a panel of 247 oncogenes and tumor suppressors, including APC. RNASeq, DNA sequencing, and clinical characteristics from 224 colon and rectal cancer samples in The Cancer Genome Atlas (TCGA) project were also obtained. Results: In the UNCseq cohort, 58 patients (83%) had at least one inactivating APC mutation, and 33 (47%) had two mutations. Of those with at least one mutation, 81% had a mutation in the MCR (residues 1281-1556), but only 5/33 (15%) with two mutations had both in the MCR. In the TCGA cohort, 162 (72%) had at least one inactivating APC mutation, and 52 (23%) had two mutations. Of those with at least one mutation, 59% had a mutation in the MCR, but only 3/52 (6%) with two mutations had both in the MCR. Gene expression of APC was higher in those with no APC mutations vs. 1-2 mutations (p = 0.015), but was not significantly different between those with one vs. two mutations (p = 0.29). The absence of APC mutations was associated with microsatellite instability (p < 0.001) and with right-sided primary tumors (p = 0.001 by chi-square). Conclusions: CRCs have high frequency of biallelic APC mutations, and the majority of tumors with APC mutations had a single mutation within the MCR region of the APC gene. These genotypic factors may impact tumor biology and clinical features.
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Yamazaki, Tomio, Akira Katsumi, Yoshihiro Okamoto, Toshio Takafuta, Shinobu Tsuzuki, Kazuo Kagami, Isamu Sugiura, Tetsuhito Kojima, Kingo Fujimura i Hidehiko Saito. "Two Distinct Novel Splice Site Mutations in a Compound Heterozygous Patient with Protein S Deficiency". Thrombosis and Haemostasis 77, nr 01 (1997): 014–20. http://dx.doi.org/10.1055/s-0038-1655729.
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SummaryGenetic analysis revealed two distinct novel splice site mutations in a compound heterozygous patient with protein S deficiency. The paternal mutation was a G-to-T transition at position -1 of the acceptor splice site of intron N (Mutation I), and the maternal mutation was a G-to-C transversion at position -1 of the donor splice site of intron C (Mutation II). Both splice site mutations decreased the mutated mRNA accumulation to the same extent, approximately 40% of the normal mRNA. However, the mutations were associated with different phenotypical expressions: the paternal mutant protein S was not detected in vivo, while the maternal mutant protein S was present in the plasma in reduced quantity. Because Mutation I caused a cryptic splicing in the mutated mRNA, resulting in a reading frameshift and premature termination, the predicted mutant protein S might be highly unstable. In contrast, Mutation II led to the substitution of Val46 by Leu, which might be much less deleterious for the synthesis, secretion and stability of the predicted mutant protein S. It was supposed that the different post-translational metabolisms produced the distinct phenotypical expressions of the mutations.
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Chattopadhyay, Maitreyi, Vera A. Stupina, Feng Gao, Christine R. Szarko, Micki M. Kuhlmann, Xuefeng Yuan, Kerong Shi i Anne E. Simon. "Requirement for Host RNA-Silencing Components and the Virus-Silencing Suppressor when Second-Site Mutations Compensate for Structural Defects in the 3′ Untranslated Region". Journal of Virology 89, nr 22 (9.09.2015): 11603–18. http://dx.doi.org/10.1128/jvi.01566-15.
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ABSTRACTTurnip crinkle virus (TCV) contains a structured 3′ region with hairpins and pseudoknots that form a complex network of noncanonical RNA:RNA interactions supporting higher-order structure critical for translation and replication. We investigated several second-site mutations in the p38 coat protein open reading frame (ORF) that arose in response to a mutation in the asymmetric loop of a critical 3′ untranslated region (UTR) hairpin that disrupts local higher-order structure. All tested second-site mutations improved accumulation of TCV in conjunction with a partial reversion of the primary mutation (TCV-rev1) but had neutral or a negative effect on wild-type (wt) TCV or TCV with the primary mutation. SHAPE (selective 2′-hydroxylacylation analyzed byprimerextension) structure probing indicated that these second-site mutations reside in an RNA domain that includes most of p38 (domain 2), and evidence for RNA:RNA interactions between domain 2 and 3′UTR-containing domain 1 was found. However, second-site mutations were not compensatory in the absence of p38, which is also the TCV silencing suppressor, or indcl-2/dcl4orago1/ago2backgrounds. One second-site mutation reduced silencing suppressor activity of p38 by altering one of two GW motifs that are required for p38 binding to double-stranded RNAs (dsRNAs) and interaction with RNA-induced silencing complex (RISC)-associated AGO1/AGO2. Another second-site mutation substantially reduced accumulation of TCV-rev1 in the absence of p38 or DCL2/DCL4. We suggest that the second-site mutations in the p38 ORF exert positive effects through a similar downstream mechanism, either by enhancing accumulation of beneficial DCL-produced viral small RNAs that positively regulate the accumulation of TCV-rev1 or by affecting the susceptibility of TCV-rev1 to RISC loaded with viral small RNAs.IMPORTANCEGenomes of positive-strand RNA viruses fold into high-order RNA structures. Viruses with mutations in regions critical for translation and replication often acquire second-site mutations that exert a positive compensatory effect through reestablishment of canonical base pairing with the altered region. In this study, two distal second-site mutations that individually arose in response to a primary mutation in a critical 3′ UTR hairpin in the genomic RNA of turnip crinkle virus did not directly interact with the primary mutation. Although different second-site changes had different attributes, compensation was dependent on the production of the viral p38 silencing suppressor and on the presence of silencing-required DCL and AGO proteins. Our results provide an unexpected connection between a 3′ UTR primary-site mutation proposed to disrupt higher-order structure and the RNA-silencing machinery.
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Wang, Zhihong, Yanhong Lin, Liping Qiu, Dezhu Zheng, Aizhen Yan, Jian Zeng i Fenghua Lan. "Hybrid minigene splicing assay verified the pathogenicity of a novel splice site variant in the dystrophin gene of a Chinese patient with typical Duchenne muscular dystrophy phenotype". Clinical Chemistry and Laboratory Medicine (CCLM) 54, nr 9 (1.09.2016): 1435–40. http://dx.doi.org/10.1515/cclm-2015-1042.
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AbstractBackground:Duchenne muscular dystrophy (DMD) is typically caused by disrupting the reading frame of the dystrophin gene: approximately 70%–80% of mutational events are represented by deletions or duplications of one or more exons in the dystrophin gene, and the remaining cases by subtle mutations, including point mutations, small indels, small inversions, and complex small rearrangements. The dystrophin gene is the largest known gene with one of the highest known rates of new mutations.Methods:Deletions and duplications were detected in theDMDgene of the proband by using multiple ligation-dependent probe amplification (MLPA). Targeted next-generation sequencing (NGS) was used in the subtle mutation detection, followed by Sanger sequencing confirmation. The effect of the mutation on the splicing of theDMDgene was assessed by bioinformatics prediction and hybrid minigene splicing assay (HMSA).Results:Neither duplication nor deletion was found in theDMDgene of the proband. While a novel splice site mutation c.6762+1G>C was identified in the proband by NGS and Sanger sequencing, and his mother was heterozygous at the same site. Bioinformatics predicted that the 5′ donor splice site of intron 46 disappeared because of the mutation, which would lead to aberrant splicing and introduce premature stop codon. The HMSA results were in agreement with the prediction.Conclusions:The novel splice site mutation caused DMD in the proband by aberrant splicing. We suggested that combined applications of MLPA, NGS, HMSA and bioinformatics are comprehensive and effective methods for diagnosis and aberrant splicing study of DMD.
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Cook, Jonathan, Elizabeth de Wolf i Nicholas Dale. "Cx26 keratitis ichthyosis deafness syndrome mutations trigger alternative splicing of Cx26 to prevent expression and cause toxicity in vitro". Royal Society Open Science 6, nr 8 (sierpień 2019): 191128. http://dx.doi.org/10.1098/rsos.191128.
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The Cx26 mRNA has not been reported to undergo alternative splicing. In expressing a series of human keratitis ichthyosis deafness (KID) syndrome mutations of Cx26 (A88V, N14K and A40V), we found the production of a truncated mRNA product. These mutations, although not creating a cryptic splice site, appeared to activate a pre-existing cryptic splice site. The alternative splicing of the mutant Cx26 mRNA could be prevented by mutating the predicted 3′, 5′ splice sites and the branch point. The presence of a C-terminal fluorescent protein tag (mCherry or Clover) was necessary for this alternative splicing to occur. Strangely, Cx26 A88V could cause the alternative splicing of co-expressed WT Cx26—suggesting a trans effect. The alternative splicing of Cx26 A88V caused cell death, and this could be prevented by the 3′, 5′ and branch point mutations. Expression of the KID syndrome mutants could be rescued by combining them with removal of the 5′ splice site. We used this strategy to enable expression of Cx26 A40V-5′ and demonstrate that this KID syndrome mutation removed CO 2 sensitivity from the Cx26 hemichannel. This is the fourth KID syndrome mutation found to abolish the CO 2 -sensitivity of the Cx26 hemichannel, and suggests that the altered CO 2 -sensitivity could contribute to the pathology of this mutation. Future research on KID syndrome mutations should take care to avoid using a C-terminal tag to track cellular localization and expression or if this is unavoidable, combine this mutation with removal of the 5′ splice site.
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Guo, Wenting, Bo Sun, John Paul Estillore, Ruiwu Wang i S. R. Wayne Chen. "The central domain of cardiac ryanodine receptor governs channel activation, regulation, and stability". Journal of Biological Chemistry 295, nr 46 (2.09.2020): 15622–35. http://dx.doi.org/10.1074/jbc.ra120.013512.
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Structural analyses identified the central domain of ryanodine receptor (RyR) as a transducer converting conformational changes in the cytoplasmic platform to the RyR gate. The central domain is also a regulatory hub encompassing the Ca2+-, ATP-, and caffeine-binding sites. However, the role of the central domain in RyR activation and regulation has yet to be defined. Here, we mutated five residues that form the Ca2+ activation site and 10 residues with negatively charged or oxygen-containing side chains near the Ca2+ activation site. We also generated eight disease-associated mutations within the central domain of RyR2. We determined the effect of these mutations on Ca2+, ATP, and caffeine activation and Mg2+ inhibition of RyR2. Mutating the Ca2+ activation site markedly reduced the sensitivity of RyR2 to Ca2+ and caffeine activation. Unexpectedly, Ca2+ activation site mutation E3848A substantially enhanced the Ca2+-independent basal activity of RyR2, suggesting that E3848A may also affect the stability of the closed state of RyR2. Mutations in the Ca2+ activation site also abolished the effect of ATP/caffeine on the Ca2+-independent basal activity, suggesting that the Ca2+ activation site is also a critical determinant of ATP/caffeine action. Mutating residues with negatively charged or oxygen-containing side chains near the Ca2+ activation site significantly altered Ca2+ and caffeine activation and reduced Mg2+ inhibition. Furthermore, disease-associated RyR2 mutations within the central domain significantly enhanced Ca2+ and caffeine activation and reduced Mg2+ inhibition. Our data demonstrate that the central domain plays an important role in channel activation, channel regulation, and closed state stability.
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Ito, Kiyoaki, Yanli Qin, Michael Guarnieri, Tamako Garcia, Karen Kwei, Masashi Mizokami, Jiming Zhang, Jisu Li, Jack R. Wands i Shuping Tong. "Impairment of Hepatitis B Virus Virion Secretion by Single-Amino-Acid Substitutions in the Small Envelope Protein and Rescue by a Novel Glycosylation Site". Journal of Virology 84, nr 24 (29.09.2010): 12850–61. http://dx.doi.org/10.1128/jvi.01499-10.
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ABSTRACT Mutations in the S region of the hepatitis B virus (HBV) envelope gene are associated with immune escape, occult infection, and resistance to therapy. We previously identified naturally occurring mutations in the S gene that alter HBV virion secretion. Here we used transcomplementation assay to confirm that the I110M, G119E, and R169P mutations in the S domain of viral envelope proteins impair virion secretion and that an M133T mutation rescues virion secretion of the I110M and G119E mutants. The G119E mutation impaired detection of secreted hepatitis B surface antigen (HBsAg), suggesting immune escape. The R169P mutant protein is defective in HBsAg secretion as well and has a dominant negative effect when it is coexpressed with wild-type envelope proteins. Although the S domain is present in all three envelope proteins, the I110M, G119E, and R169P mutations impair virion secretion through the small envelope protein. Conversely, coexpression of just the small envelope protein of the M133T mutant could rescue virion secretion. The M133T mutation could also overcome the secretion defect caused by the G145R immune-escape mutation or mutation at N146, the site of N-linked glycosylation. In fact, the M133T mutation creates a novel N-linked glycosylation site (131NST133). Destroying this site by N131Q/T mutation or preventing glycosylation by tunicamycin treatment of transfected cells abrogated the effect of the M133T mutation. Our findings demonstrate that N-linked glycosylation of HBV envelope proteins is critical for virion secretion and that the secretion defect caused by mutations in the S protein can be rescued by an extra glycosylation site.
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Bebenek, Anna, Geraldine T. Carver, Holly Kloos Dressman, Farid A. Kadyrov, Joseph K. Haseman, Vasiliy Petrov, William H. Konigsberg, Jim D. Karam i John W. Drake. "Dissecting the Fidelity of Bacteriophage RB69 DNA Polymerase: Site-Specific Modulation of Fidelity by Polymerase Accessory Proteins". Genetics 162, nr 3 (1.11.2002): 1003–18. http://dx.doi.org/10.1093/genetics/162.3.1003.
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Abstract Bacteriophage RB69 encodes a replicative B-family DNA polymerase (RB69 gp43) with an associated proofreading 3′ exonuclease. Crystal structures have been determined for this enzyme with and without DNA substrates. We previously described the mutation rates and kinds of mutations produced in vivo by the wild-type (Pol+ Exo+) enzyme, an exonuclease-deficient mutator variant (Pol+ Exo-), mutator variants with substitutions at Tyr567 in the polymerase active site (PolM Exo+), and the double mutator PolM Exo-. Comparing the mutational spectra of the Pol+ Exo- and Pol+ Exo+ enzymes revealed the patterns and efficiencies of proofreading, while Tyr567 was identified as an important determinant of base-selection fidelity. Here, we sought to determine how well the fidelities of the same enzymes are reflected in vitro. Compared to their behavior in vivo, the three mutator polymerases exhibited modestly higher mutation rates in vitro and their mutational predilections were also somewhat different. Although the RB69 gp43 accessory proteins exerted little or no effect on total mutation rates in vitro, they strongly affected mutation rates at many specific sites, increasing some rates and decreasing others.
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Yu, Yongfeng, Rongrong Chen, Jun Zhao, Xin Yi i Shun Lu. "Analysis of canonical and noncanonical splicing site mutation of MET that causes exon 14 skipping." Journal of Clinical Oncology 38, nr 15_suppl (20.05.2020): e21513-e21513. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.e21513.
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e21513 Background: The hepatocyte growth factor receptor gene ( MET) exon 14 skipping ( METex14) has been wildly accepted as a driver alteration in lung cancer targetable by tyrosine kinase inhibitors (TKIs) such as crizotinib. While it is easy to interpret canonical splicing site mutations, it is more controversial to interpret noncanonical splicing site mutations. Methods: Hybrid capture–based next generation sequencing of 59-1021 genes including MET was performed at the request of individual treating physicians. The mutation profiling of MET were retrospectively analyzed. Results: Of 3500 lung cancers profiled by comprehensive genomic profiling, 211 harbored MET single nucleotide mutation (SNV), or small insertion/deletion (InDel). Among which 66 cases were predicted to have METex14. Forty-two (62.1%) cases harbored mutations affecting the splicing donor site, while 24 harbored mutations affecting the splicing acceptor site. Thirty-four of the 42 cases affecting the splicing donor site had mutations in the canonical splicing site, including 3 cases harbored MET c.3028G > A/T/C mutation, 14 cases harbored c.3028+1G > A/T/C, 10 cases harbored c.3028+2T > A/C/G, 8 cases harbored deletion of the whole canonical splicing site. Interestingly, all the seven cases affecting the noncanonical splicing donor site were c.3028+3A > G/T mutation. On the contrary, all the 24 mutations affecting the splicing acceptor site were deletion with/without insertion, with 12 affecting the canonical site and 12 the noncanonical site. We speculated that the polypyrimidine tract around the splicing acceptor site making it more susceptible to deletion/insertion, and thus single nucleotide variant less common. Conclusions: At the 5’ end of the exon 14 of MET, most of the genome alterations are deletions with/without insertions affecting the canonical and noncanonical splicing acceptor site. At the 3’ end, they are mostly point mutations affecting the splice donor site. The noncanonical splicing donor site c.3028+3A plays crucial role in the splicing of exon 14 in MET.
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Claes, Kathleen, Eva Machackova, Michel De Vos, Bruce Poppe, Anne De Paepe i Ludwine Messiaen. "Mutation Analysis of the BRCA1 and BRCA2 Genes in the Belgian Patient Population and Identification of a Belgian Founder Mutation BRCA1 IVS5+3A>G". Disease Markers 15, nr 1-3 (1999): 69–73. http://dx.doi.org/10.1155/1999/241046.
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Since the identification of the BRCA1 and BRCA2 genes, several hundred different germline mutations in both genes have been reported. Recurrent mutations are rare and mainly due to founder effects. As the mutational spectrum of the BRCA1 and BRCA2 genes in the Belgian patient population is largely unknown, we initiated mutation analysis for the complete coding sequence of both genes in Belgian families with multiple breast and/or ovarian cancer patients and in “sporadic” patients with early onset disease. We completed the analysis in 49 families and in 19 “sporadic” female patients with early onset breast and/or ovarian cancer. In 15 families we identified a mutation (12 mutations in BRCA1 and 3 mutations in BRCA2). In 5 apparently unrelated families the same splice site mutation was identified (BRCA1 IVS5+3A>G). Haplotype analysis revealed a common haplotype immediately flanking the mutation in all families suggesting that disease alleles are identical by descent. In none of the 19 sporadic patients was a mutation found.
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Jenkins, Gareth J. S., Morteza Hashemzadeh Chaleshtori, Honglin Song i James M. Parry. "Mutation analysis using the restriction site mutation (RSM) assay". Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 405, nr 2 (wrzesień 1998): 209–20. http://dx.doi.org/10.1016/s0027-5107(98)00138-9.
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Lin, Wen-Ying, Kang-Yang Jih i Tzyh-Chang Hwang. "A single amino acid substitution in CFTR converts ATP to an inhibitory ligand". Journal of General Physiology 144, nr 4 (15.09.2014): 311–20. http://dx.doi.org/10.1085/jgp.201411247.
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Cystic fibrosis (CF), one of the most common lethal genetic diseases, is caused by loss-of-function mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel that, when phosphorylated, is gated by ATP. The third most common pathogenic mutation, a glycine-to-aspartate mutation at position 551 or G551D, shows a significantly decreased open probability (Po) caused by failure of the mutant channel to respond to ATP. Recently, a CFTR-targeted drug, VX-770 (Ivacaftor), which potentiates G551D-CFTR function in vitro by boosting its Po, has been approved by the FDA to treat CF patients carrying this mutation. Here, we show that, in the presence of VX-770, G551D-CFTR becomes responsive to ATP, albeit with an unusual time course. In marked contrast to wild-type channels, which are stimulated by ATP, sudden removal of ATP in excised inside-out patches elicits an initial increase in macroscopic G551D-CFTR current followed by a slow decrease. Furthermore, decreasing [ATP] from 2 mM to 20 µM resulted in a paradoxical increase in G551D-CFTR current. These results suggest that the two ATP-binding sites in the G551D mutant mediate opposite effects on channel gating. We introduced mutations that specifically alter ATP-binding affinity in either nucleotide-binding domain (NBD1 or NBD2) into the G551D background and determined that this disease-associated mutation converts site 2, formed by the head subdomain of NBD2 and the tail subdomain of NBD1, into an inhibitory site, whereas site 1 remains stimulatory. G551E, but not G551K or G551S, exhibits a similar phenotype, indicating that electrostatic repulsion between the negatively charged side chain of aspartate and the γ-phosphate of ATP accounts for the observed mutational effects. Understanding the molecular mechanism of this gating defect lays a foundation for rational drug design for the treatment of CF.
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Puranen, T. J., M. H. Poutanen, H. E. Peltoketo, P. T. Vihko i R. K. Vihko. "Site-directed mutagenesis of the putative active site of human 17β-hydroxysteroid dehydrogenase type 1". Biochemical Journal 304, nr 1 (15.11.1994): 289–93. http://dx.doi.org/10.1042/bj3040289.
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Several amino acid residues (Cys54, Tyr155, His210, His213 and His221) at a putative catalytic site of human 17 beta-hydroxysteroid dehydrogenase type 1 were mutated to Ala. Replacement of His221 by Ala remarkably reduced the catalytic activity, which resulted from a change of both the Km and the Vmax. values of the enzyme. Compared with the wild-type enzyme, the catalytic efficiency of the His221-->Ala mutant was reduced 20-fold for the oxidative reaction and 11-fold for the reductive reaction. With similar mutations at His210 or His213, no notable effects on the catalytic properties of the enzyme were detected. However, a simultaneous mutation of these amino acid residues decreased the Vmax. values of both oxidation and reduction by about 50% from those measured for the wild-type enzyme. Although Cys54 has been localized in the cofactor-binding region of the enzyme, a Cys54-->Ala mutation did not lead to changes in the enzymic activity. The most dramatic effects on the catalytic properties of the enzyme were achieved by mutating Tyr155, which resulted in an almost completely inactivation of the enzyme. The decreased enzymic activities of the Tyr155-->Ala, His210-->Ala + His213-->Ala and His221-->Ala mutations were also reflected in a reduced immunoreactivity of the enzymes. The results thus suggest that the lower catalytic efficiency of the mutant enzymes is due to an exchange of catalytically important amino acid residues and/or remarkable alterations in the three-dimensional structure of the enzyme. The recently detected polymorphisms (Ala237<-->Val and Ser312<-->Gly) were not found to affect either the catalytic or the immunological properties of the type 1 enzyme.
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Neinavaie, Fargam, i Andrew Kramer. "Abstract A038: Does mutation rate of cancer cells change as the stage of the disease advances?" Cancer Research 82, nr 10_Supplement (15.05.2022): A038. http://dx.doi.org/10.1158/1538-7445.evodyn22-a038.
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Abstract Tumorigenesis begins with cells that have normal mutation rates. As the cells become immortal and accumulate different numbers of mutations along the way, current literature splits in two describing if and how mutation rates increase as the cancer progresses. Some argue that as the mutation rates are small (mutation rate in healthy cells is per nucleotide site per cell division) and malignant cells are loaded with thousands of different types of mutations, they must have acquired a mutator phenotype early in their development. Others argue that immortality of malignant cells, and their rate of proliferation is responsible for their genetic instability and number of mutations observed in their genome. We conduct a review of the literature with the key words “mutation rate” and “cancer” in their title. We review 123 papers and found that description of mutation rates varied in the published work. This leads to estimates of mutation rates in cancer from per nucleotide site per cell division. Some of this variation in mutational rates arises across different organs of the body, for instance liver cells have a higher mutation rate than heart and brain. Cancer treatments such as chemotherapy influence mutation rate, and mutation rate of drug and multidrug resistance cancer cell is higher than newly diagnosed cancer cells. Further, mutation rate is defined differently in different literature, some papers measure the mutation rate in a population or a gene, some calculate it per base pair, genome, or mitosis. Here we provide a comprehensive picture of published mutation rates across different cancer types in order to inform cancer models and treatment plans for individual patients. We propose a unified framework for discussing and reporting mutation rate of cancer cells and formulations on how to switch between definitions. Citation Format: Fargam Neinavaie, Andrew Kramer. Does mutation rate of cancer cells change as the stage of the disease advances? [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr A038.
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Yang, Zhi, Priyatama Pandey, Darryl Shibata, David V. Conti, Paul Marjoram i Kimberly D. Siegmund. "HiLDA: a statistical approach to investigate differences in mutational signatures". PeerJ 7 (28.08.2019): e7557. http://dx.doi.org/10.7717/peerj.7557.
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We propose a hierarchical latent Dirichlet allocation model (HiLDA) for characterizing somatic mutation data in cancer. The method allows us to infer mutational patterns and their relative frequencies in a set of tumor mutational catalogs and to compare the estimated frequencies between tumor sets. We apply our method to two datasets, one containing somatic mutations in colon cancer by the time of occurrence, before or after tumor initiation, and the second containing somatic mutations in esophageal cancer by sex, age, smoking status, and tumor site. In colon cancer, the relative frequencies of mutational patterns were found significantly associated with the time of occurrence of mutations. In esophageal cancer, the relative frequencies were significantly associated with the tumor site. Our novel method provides higher statistical power for detecting differences in mutational signatures.
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Kraut, Daniel A., Paul A. Sigala, Timothy D. Fenn i Daniel Herschlag. "Dissecting the paradoxical effects of hydrogen bond mutations in the ketosteroid isomerase oxyanion hole". Proceedings of the National Academy of Sciences 107, nr 5 (11.01.2010): 1960–65. http://dx.doi.org/10.1073/pnas.0911168107.
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The catalytic importance of enzyme active-site interactions is frequently assessed by mutating specific residues and measuring the resulting rate reductions. This approach has been used in bacterial ketosteroid isomerase to probe the energetic importance of active-site hydrogen bonds donated to the dienolate reaction intermediate. The conservative Tyr16Phe mutation impairs catalysis by 105-fold, far larger than the effects of hydrogen bond mutations in other enzymes. However, the less-conservative Tyr16Ser mutation, which also perturbs the Tyr16 hydrogen bond, results in a less-severe 102-fold rate reduction. To understand the paradoxical effects of these mutations and clarify the energetic importance of the Tyr16 hydrogen bond, we have determined the 1.6-Å resolution x-ray structure of the intermediate analogue, equilenin, bound to the Tyr16Ser mutant and measured the rate effects of mutating Tyr16 to Ser, Thr, Ala, and Gly. The nearly identical 200-fold rate reductions of these mutations, together with the 6.4-Å distance observed between the Ser16 hydroxyl and equilenin oxygens in the x-ray structure, strongly suggest that the more moderate rate effect of this mutant is not due to maintenance of a hydrogen bond from Ser at position 16. These results, additional spectroscopic observations, and prior structural studies suggest that the Tyr16Phe mutation results in unfavorable interactions with the dienolate intermediate beyond loss of a hydrogen bond, thereby exaggerating the apparent energetic benefit of the Tyr16 hydrogen bond relative to the solution reaction. These results underscore the complex energetics of hydrogen bonding interactions and site-directed mutagenesis experiments.
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Riedmayr, Lisa M., Sybille Böhm, Martin Biel i Elvir Becirovic. "Enigmatic rhodopsin mutation creates an exceptionally strong splice acceptor site". Human Molecular Genetics 29, nr 2 (9.12.2019): 295–304. http://dx.doi.org/10.1093/hmg/ddz291.
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Abstract The c.620 T > G mutation in rhodopsin found in the first mapped autosomal dominant retinitis pigmentosa (adRP) locus is associated with severe, early-onset RP. Intriguingly, another mutation affecting the same nucleotide (c.620 T > A) is related to a mild, late-onset RP. Assuming that both mutations are missense mutations (Met207Arg and Met207Lys) hampering the ligand-binding pocket, previous work addressed how they might differentially impair rhodopsin function. Here, we investigated the impact of both mutations at the mRNA and protein level in HEK293 cells and in the mouse retina. We show that, in contrast to c.620 T > A, c.620 T > G is a splicing mutation, which generates an exceptionally strong splice acceptor site (SAS) resulting in a 90 bp in-frame deletion and protein mislocalization in vitro and in vivo. Moreover, we identified the core element underlying the c.620 T > G SAS strength. Finally, we demonstrate that the c.620 T > G SAS is very flexible in branch point choice, which might explain its remarkable performance. Based on these results, we suggest that (i) point mutations should be routinely tested for mRNA splicing to avoid dispensable analysis of mutations on protein level, which do not naturally exist. (ii) Puzzling disease courses of mutations in other genes might also correlate with their effects on mRNA splicing. (iii) Flexibility in branch point choice might be another factor influencing the SAS strength. (iv) The core splice element identified in this study could be useful for biotechnological applications requiring effective SAS.
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Russo, Antonio, Viviana Bazan, Barry Iacopetta, David Kerr, Thierry Soussi i Nicola Gebbia. "The TP53 Colorectal Cancer International Collaborative Study on the Prognostic and Predictive Significance of p53 Mutation: Influence of Tumor Site, Type of Mutation, and Adjuvant Treatment". Journal of Clinical Oncology 23, nr 30 (20.10.2005): 7518–28. http://dx.doi.org/10.1200/jco.2005.00.471.
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Purpose The aims of the TP53 Colorectal Cancer (CRC) International Collaborative Study were to evaluate the possible associations between specific TP53 mutations and tumor site, and to evaluate the prognostic and predictive significance of these mutations in different site, stage, and treatment subgroups. Patients and Methods A total of 3,583 CRC patients from 25 different research groups in 17 countries were recruited to the study. Patients were divided into three groups according to site of the primary tumor. TP53 mutational analyses spanned exons 4 to 8. Results TP53 mutations were found in 34% of the proximal colon tumors and in 45% of the distal colon and rectal tumors. They were associated with lymphatic invasion in proximal tumors. In distal colon tumors, deletions causing loss of amino acids were associated with worse survival. In proximal colon tumors, mutations in exon 5 showed a trend toward statistical significance (P < .05) when overall survival was considered. Dukes' C tumors with wild-type TP53 and those with mutated TP53 (proximal tumors) showed significantly better prognosis when treated with adjuvant chemotherapy. Conclusion Analysis of TP53 mutations from a large cohort of CRC patients has identified tumor site, type of mutation, and adjuvant treatment as important factors in determining the prognostic significance of this genetic alteration.
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Doward, W., R. Perveen, I. C. Lloyd, A. E. A. Ridgway, L. Wilson i G. C. M. Black. "A mutation in the RIEG1 gene associated with Peters’ anomaly". Journal of Medical Genetics 36, nr 2 (1.02.1999): 152–55. http://dx.doi.org/10.1136/jmg.36.2.152.
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Mutations within the RIEG1 homeobox gene on chromosome 4q25 have previously been reported in association with Rieger syndrome. We report a 3′ splice site mutation within the 3rd intron of the RIEG1 gene which is associated with unilateral Peters’ anomaly. The mutation is a single base substition of A to T at the invariant -2 site of the 3′ splice site. Peters’ anomaly, which is characterised by ocular anterior segment dysgenesis and central corneal opacification, is distinct from Rieger anomaly. This is the first description of a RIEG1 mutation associated with Peters’ anomaly.
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Park, S., B. Park, I. Hwang, S. Lee, E. Cho, W. Kang, J. Ahn, M. Ahn i K. Park. "Comparison of the epidermal growth factor receptor gene mutation in matched primary tumor and lymph node metastasis of non-small cell lung cancer". Journal of Clinical Oncology 25, nr 18_suppl (20.06.2007): 7614. http://dx.doi.org/10.1200/jco.2007.25.18_suppl.7614.
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7614 Background: Mutations in epidermal growth factor receptor (EGFR) are considered as a strong predictive marker to EGFR tyrosine kinase inhibitors (TKIs) in non-small-cell lung cancer (NSCLC). Recent studies suggested EGFR status may change between primary NSCLC and corresponding metastatic site. However, it has not fully been evaluated whether EGFR mutation differs in metastases compared to primary NSCLC. Methods: In total, 128 tumor samples from 64 NSCLC patients were investigated comparing matched 64 primary tumors, and 64 lymph node metastases. The epidermal growth factor receptor mutation status was analyzed by a direct sequencing method (exons 18–21 in EGFR) on tumor samples of primary NSCLC and corresponding lymph node metastasis. Results: In 17.2% of patents (11/64), EGFR mutation was identified in either primary NSCLC or metastasis by DNA sequencing. Six (54.5%) out of eleven cases showed discordance of EGFR mutation in the primary tumor/metastasis site. Two cases showed EGFR mutation in the metastasis but not in the primary tumor, while, in four cases, EGFR mutation was detected in the primary tumor but not in the metastasis site. The majority of discordance of EGFR mutations was identified in exon 19 (83.3%, 5/6). The median overall survival (OS) was 17.7 months (95% confidence interval, 9.4–20.0). Median OS was not varied by the discordance of EGFR mutation status between primary NSCLC and corresponding metastatic site. Conclusions: The status of EGFR mutation in primary NSCLC and that in corresponding metastasis site varied in considerable cases by DNA sequencing. Whether the status of EGFR mutation changes during the process of metastasis remains to be evaluated. Future study to evaluate the correlation of tumour response to TKIs and the discordance of the EGFR mutation status is warranted. No significant financial relationships to disclose.
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Joerger, Andreas C., Hwee Ching Ang, Dmitry B. Veprintsev, Caroline M. Blair i Alan R. Fersht. "Structures of p53 Cancer Mutants and Mechanism of Rescue by Second-site Suppressor Mutations". Journal of Biological Chemistry 280, nr 16 (9.02.2005): 16030–37. http://dx.doi.org/10.1074/jbc.m500179200.
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We have solved the crystal structures of three oncogenic mutants of the core domain of the human tumor suppressor p53. The mutations were introduced into a stabilized variant. The cancer hot spot mutation R273H simply removes an arginine involved in DNA binding without causing structural distortions in neighboring residues. In contrast, the “structural” oncogenic mutations H168R and R249S induce substantial structural perturbation around the mutation site in the L2 and L3 loops, respectively. H168R is a specific intragenic suppressor mutation for R249S. When both cancer mutations are combined in the same molecule, Arg168mimics the role of Arg249in wild type, and the wild type conformation is largely restored in both loops. Our structural and biophysical data provide compelling evidence for the mechanism of rescue of mutant p53 by intragenic suppressor mutations and reveal features by which proteins can adapt to deleterious mutations.
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Moir, Robyn D., Karen V. Puglia i Ian M. Willis. "A Gain-of-Function Mutation in the Second Tetratricopeptide Repeat of TFIIIC131 Relieves Autoinhibition of Brf1 Binding". Molecular and Cellular Biology 22, nr 17 (1.09.2002): 6131–41. http://dx.doi.org/10.1128/mcb.22.17.6131-6141.2002.
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ABSTRACT The interaction between the tetratricopeptide repeat (TPR)-containing subunit of TFIIIC, TFIIIC131, and the TFIIB-related factor Brf1 represents a limiting step in the assembly of the RNA polymerase III (pol III) initiation factor TFIIIB. This assembly reaction is facilitated by dominant mutations that map in and around TPR2. Structural modeling of TPR1 to TPR3 from TFIIIC131 shows that one such mutation, PCF1-2, alters a residue in the ligand-binding groove of the TPR superhelix whereas another mutation, PCF1-1, changes a surface-accessible residue on the back side of the TPR superhelix. In this work, we show that the PCF1-1 mutation (H190Y) increases the binding affinity for Brf1, but does not affect the binding affinity for Bdp1, in the TFIIIC-dependent assembly of TFIIIB. Interestingly, binding studies with TFIIIC131 fragments indicate that Brf1 does not interact directly at the site of the PCF1-1 mutation. Rather, the data suggest that the mutation overcomes the previously documented autoinhibition of Brf1 binding. These findings together with the results from site-directed mutagenesis support the hypothesis that gain-of-function mutations at amino acid 190 in TPR2 stabilize an alternative conformation of TFIIIC131 that promotes its interaction with Brf1.
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Mandl, Christian W., Steven L. Allison, Heidemarie Holzmann, Tamara Meixner i Franz X. Heinz. "Attenuation of Tick-Borne Encephalitis Virus by Structure-Based Site-Specific Mutagenesis of a Putative Flavivirus Receptor Binding Site". Journal of Virology 74, nr 20 (15.10.2000): 9601–9. http://dx.doi.org/10.1128/jvi.74.20.9601-9609.2000.
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ABSTRACT The impact of a specific region of the envelope protein E of tick-borne encephalitis (TBE) virus on the biology of this virus was investigated by a site-directed mutagenesis approach. The four amino acid residues that were analyzed in detail (E308 to E311) are located on the upper-lateral surface of domain III according to the X-ray structure of the TBE virus protein E and are part of an area that is considered to be a potential receptor binding determinant of flaviviruses. Mutants containing single amino acid substitutions, as well as combinations of mutations, were constructed and analyzed for their virulence in mice, growth properties in cultured cells, and genetic stability. The most significant attenuation in mice was achieved by mutagenesis of threonine 310. Combining this mutation with deletion mutations in the 3′-noncoding region yielded mutants that were highly attenuated. The biological effects of mutation Thr 310 to Lys, however, could be reversed to a large degree by a mutation at a neighboring position (Lys 311 to Glu) that arose spontaneously during infection of a mouse. Mutagenesis of the other positions provided evidence for the functional importance of residue 308 (Asp) and its charge interaction with residue 311 (Lys), whereas residue 309 could be altered or even deleted without any notable consequences. Deletion of residue 309 was accompanied by a spontaneous second-site mutation (Phe to Tyr) at position 332, which in the three-dimensional structure of protein E is spatially close to residue 309. The information obtained in this study is relevant for the development of specific attenuated flavivirus strains that may serve as future live vaccines.
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INVERNIZZI, Cédric, Jonathan IMHOF, Gabriela BURKARD, Katharina SCHMID i Arminio BOSCHETTI. "Effects of mutations at the two processing sites of the precursor for the small subunit of ribulose-bisphosphate carboxylase in Chlamydomonas reinhardtii". Biochemical Journal 366, nr 3 (15.09.2002): 989–98. http://dx.doi.org/10.1042/bj20020378.
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The role of the two processing sites in the precursor of the small subunit (SS) of ribulose-1,5-bisphosphate carboxylase/oxygenase (pSS) of Chlamydomonas reinhardtii was studied by introducing mutations at the cleavage sites for the stromal processing peptidases SPP-1 and SPP-2, which hydrolyse wild-type pSS (20.6kDa) to an intermediate-sized product iSS (18.3kDa) and to the mature SS (16.3kDa), respectively. The mutations introduced into cDNA resulted in exchange of (a) two amino acids flanking processing site 1, or (b) one or (c) both amino acids flanking processing site 2. Mutation (a) prevented pSS from being processed at site 1 but not from cleavage at site 2. Mutation (c) abolished the action of SPP-2 but not SPP-1. When pSS with mutation (c) was imported into isolated chloroplasts, iSS accumulated while SS formation was abolished. However, mature SS was produced even in the absence of iSS synthesis (mutation a). Import of pSS bearing mutation (b), which only partially inhibited processing at the SPP-2 site, slowed the rate of SS formation down whereas iSS and some slightly smaller derivatives accumulated. These experiments suggested that in Chlamydomonas processing of pSS can occur in two steps, whereby the first step is facultative. The same three mutations were studied in vivo after transformation of SS-deficient C. reinhardtii T60-3 with mutated genomic DNA. Growth and photosynthesis was as in control transformants, except for the slower-growing transformants (mutation c) where no mature SS was immuno-detected. However, pSS fragments with molecular masses between those of iSS and SS were present even in the ribulose-1,5-bisphosphate carboxylase/oxygenase holoenzyme.
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Ichikawa, Shoji, Kenneth W. Lyles i Michael J. Econs. "A Novel GALNT3 Mutation in a Pseudoautosomal Dominant Form of Tumoral Calcinosis: Evidence That the Disorder Is Autosomal Recessive". Journal of Clinical Endocrinology & Metabolism 90, nr 4 (1.04.2005): 2420–23. http://dx.doi.org/10.1210/jc.2004-2302.
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Abstract Familial tumoral calcinosis is a rare metabolic disorder, characterized by ectopic calcification and hyperphosphatemia. Recently biallelic mutations in the GalNAc transferase 3 (GALNT3) gene were identified in two families with tumoral calcinosis. In the present study, we performed mutation analysis of the GALNT3 gene in a multigenerational family, which was originally described to have an autosomal dominant form of tumoral calcinosis. We identified a novel splice site mutation in intron 1 (IVS1–2a→t), likely leading to skipping of exon 2. The proband was a compound heterozygote for the splice site mutation and the previously reported nonsense mutation (484C→T; R162X). His affected maternal great uncle was homozygous for the splice site mutation. Biallelic mutations found in two generations demonstrated that the family had pseudoautosomal dominant inheritance, confirming that tumoral calcinosis is in fact an autosomal recessive trait. However, genetic and biochemical findings suggest that carriers of a single mutation may also manifest subtle biochemical abnormalities. Furthermore, coexpression of GALNT3 and fibroblast growth factor 23 (FGF23), a key regulator of phosphate homeostasis, in certain tissues suggests that O-glycosylation of FGF23 by GALNT3 may be necessary for proper function of FGF23.
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Rodriguez, Cesar, Joshua Tompkin, Jill Hazel i Patricia L. Foster. "Induction of a DNA Nickase in the Presence of Its Target Site Stimulates Adaptive Mutation in Escherichia coli". Journal of Bacteriology 184, nr 20 (15.10.2002): 5599–608. http://dx.doi.org/10.1128/jb.184.20.5599-5608.2002.
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ABSTRACT Adaptive mutation to Lac+ in Escherichia coli strain FC40 depends on recombination functions and is enhanced by the expression of conjugal functions. To test the hypothesis that the conjugal function that is important for adaptive mutation is the production of a single-strand nick at the conjugal origin, we supplied an exogenous nicking enzyme, the gene II protein (gIIp) of bacteriophage f1, and placed its target sequence near the lac allele. When both gIIp and its target site were present, adaptive mutation was stimulated three- to fourfold. Like normal adaptive mutations, gIIp-induced mutations were recA+ and ruvC+ dependent and were mainly single-base deletions in runs of iterated bases. In addition, gIIp with its target site could substitute for conjugal functions in adaptive mutation. These results support the hypothesis that nicking at the conjugal origin initiates the recombination that produces adaptive mutations in this strain of E. coli, and they suggest that nicking may be the only conjugal function required for adaptive mutation.
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Elliott, Steve, Tony Lorenzini, David Chang, Jack Barzilay i Evelyne Delorme. "Mapping of the Active Site of Recombinant Human Erythropoietin". Blood 89, nr 2 (15.01.1997): 493–502. http://dx.doi.org/10.1182/blood.v89.2.493.
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Abstract Recombinant human erythropoietin (rHuEPO) variants have been constructed to identify amino acid residues important for biological activity. Immunoassays were used to determine the effect of each mutation on rHuEPO folding. With this strategy, we could distinguish between mutations that affected bioactivity directly and those that affected bioactivity because the mutation altered rHuEPO conformation. Four regions were found to be important for bioactivity: amino acids 11 to 15, 44 to 51, 100 to 108, and 147 to 151. EPO variants could be divided into two groups according to the differential effects on EPO receptor binding activity and in vitro biologic activity. This suggests that rHuEPO has two separate receptor binding sites. Mutations in basic residues reduced the biologic activity, whereas mutations in acidic residues did not. This suggests that electrostatic interactions between rHuEPO and the human EPO receptor may involve positive charges on rHuEPO.
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Van Kuilenburg, André B. P., Rutger Meinsma, Eva Beke, Barbara Bobba, Patrizia Boffi, Gregory M. Enns, David R. Witt i Doreen Dobritzsch. "Identification of three novel mutations in the dihydropyrimidine dehydrogenase gene associated with altered pre-mRNA splicing or protein function". Biological Chemistry 386, nr 4 (1.04.2005): 319–24. http://dx.doi.org/10.1515/bc.2005.038.
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AbstractDihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of the pyrimidine bases uracil and thymine, as well as of the widely used chemotherapeutic drug 5-fluorouracil (5FU). Analysis of the DPD gene (DPYD) in two patients presenting with complete DPD deficiency and the parents of an affected child showed the presence of three novel mutations, including one splice site mutation IVS11+1G→T and the missense mutations 731A→C (E244V) and 1651G→A (A551T). The G→T mutation in the invariant GT splice donor site flanking exon 11 (IVS11+1G→T) created a cryptic splice site within exon 11. As a consequence, a 141-bp fragment encoding the aminoacid residues 400–446 of the primary sequence of the DPD protein was missing in the mature DPD mRNA. Analysis of the crystal structure of pig DPD suggested that the E244V mutation might interfere with the electron flow between NADPH and the pyrimidine binding site of DPD. The A551T point mutation might prevent binding of the prosthetic group FMN and affect folding of the DPD protein. The identification of these novel mutations inDPYDwill allow the identification of patients with an increased risk of developing severe 5FU-associated toxicity.
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Turner, Jeremy J. O., Poloko D. Leotlela, Anna A. J. Pannett, Simon A. Forbes, J. H. Duncan Bassett, Brian Harding, Paul T. Christie i in. "Frequent Occurrence of an Intron 4 Mutation in Multiple Endocrine Neoplasia Type 1". Journal of Clinical Endocrinology & Metabolism 87, nr 6 (1.06.2002): 2688–93. http://dx.doi.org/10.1210/jcem.87.6.8607.
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MEN1 is an autosomal dominant disorder characterized by parathyroid, pituitary, and pancreatic tumors. The MEN1 gene is located on chromosome 11q13 and encodes a 610-amino acid protein. MEN1 mutations are of diverse types and are scattered throughout the coding region, such that almost every MEN1 family will have its individual mutation. To further characterize such mutations we ascertained 34 unrelated MEN1 probands and undertook DNA sequence analysis. This identified 17 different mutations in 24 probands (2 nonsense, 2 missense, 2 in-frame deletions, 5 frameshift deletions, 1 frameshift deletional-insertion, 3 frameshift insertions, 1 donor splice site mutation, and a g→a transition that resulted in a novel acceptor splice site in intron 4). The intron 4 mutation was found in 7 unrelated families, and the tumors in these families varied considerably, indicating a lack of genotype-phenotype correlation. However, this intron 4 mutation is the most frequently occurring germline MEN1 mutation (∼10% of all mutations), and together with 5 others at codons 83–84, 118–119, 209–211, 418, and 516, accounts for 36.6% of all mutations, a finding that indicates an approach for identifying the widely diverse MEN1 mutations.
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Amano, Eiichiro, Tomokatsu Yoshida, Ikuko Mizuta, Jun Oyama, Shingo Sakashita, Syunsuke Ueyama, Akira Machida i Takanori Yokota. "Activation of a Cryptic Splice Site of GFAP in a Patient With Adult-Onset Alexander Disease". Neurology Genetics 7, nr 6 (1.10.2021): e626. http://dx.doi.org/10.1212/nxg.0000000000000626.
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Background and ObjectiveAlexander disease (ALXDRD) is an autosomal dominant neurologic disorder caused by mutations in the glial fibrillary acidic protein (GFAP) gene and is pathologically defined by Rosenthal fiber accumulation. Most mutations are exonic missense mutations, and splice site mutations are rare. We report a very-late-onset autopsied case of adult-onset ALXDRD with a novel splice site mutation.MethodsGenetic testing of GFAP was performed by Sanger sequencing. Using autopsied brain tissues, GFAP transcript analysis was performed.ResultsThe patient presented mild upper motor neuron symptoms in contrast to the severe atrophy of spinal cord and medulla oblongata. The patient had c.619-1G>A mutation, which is located in the canonical splice acceptor site of intron 3. The brain RNA analysis identified the r.619_621del (p.Glu207del) mutation, which is explained by the activation of the cryptic splice acceptor site in the second and third nucleotides from the 5′ end of the exon 4.DiscussionGFAP gene expression analysis is necessary to clarify the effects of intronic mutations on splicing, even if they are in canonical splice sites. This case showed a much milder phenotype than those in previous cases with missense mutations at Glu207, thereby expanding the clinical spectrum of ALXDRD with Glu207 mutation.
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Martyn, Gabriella E., Beeke Wienert, Ryo Kurita, Yukio Nakamura, Kate G. R. Quinlan i Merlin Crossley. "A natural regulatory mutation in the proximal promoter elevates fetal globin expression by creating a de novo GATA1 site". Blood 133, nr 8 (21.02.2019): 852–56. http://dx.doi.org/10.1182/blood-2018-07-863951.
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Abstract β-hemoglobinopathies, such as sickle cell disease and β-thalassemia, result from mutations in the adult β-globin gene. Reactivating the developmentally silenced fetal γ-globin gene elevates fetal hemoglobin levels and ameliorates symptoms of β-hemoglobinopathies. The continued expression of fetal γ-globin into adulthood occurs naturally in a genetic condition termed hereditary persistence of fetal hemoglobin (HPFH). Point mutations in the fetal γ-globin proximal promoter can cause HPFH. The −113A>G HPFH mutation falls within the −115 cluster of HPFH mutations, a binding site for the fetal globin repressor BCL11A. We demonstrate that the −113A>G HPFH mutation, unlike other mutations in the cluster, does not disrupt BCL11A binding but rather creates a de novo binding site for the transcriptional activator GATA1. Introduction of the −113A>G HPFH mutation into erythroid cells using the clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) system increases GATA1 binding and elevates fetal globin levels. These results reveal the mechanism by which the −113A>G HPFH mutation elevates fetal globin and demonstrate the sensitivity of the fetal globin promoter to point mutations that often disrupt repressor binding sites but here create a de novo site for an erythroid activator.
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Vora, Hemangini H., Shalvi V. Mehta, Shilin N. Shukla i Pankaj M. Shah. "No Mutation Detected in Five Hot Spot Codons of the Tp53 Gene by Restriction Site Mutation Analysis in Patients with Carcinoma of the Tongue". International Journal of Biological Markers 25, nr 1 (styczeń 2010): 46–51. http://dx.doi.org/10.1177/172460081002500107.
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The present study evaluated 5 of the 8 main TP53 mutation hot spots in cancer by restriction site mutation analysis and compared the results with p53 protein expression in patients with cancer of the tongue. Tumor samples from 49 patients with tongue cancer were screened for TP53 mutations in exons 5 through 8 by PCR restriction site mutation analysis and for p53 protein expression by immunohistochemistry using the DO-7 antibody. Nuclear accumulation of p53 protein was seen in 22% (11/49) of the tumors, whereas none of the patients exhibited TP53 mutations in exons 5 through 8. The observed data suggest that TP53 mutations alone are not responsible for abnormal accumulation of p53 protein in tobacco-chewing-mediated tongue carcinogenesis.
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Morris, Van Karlyle, Michael J. Overman, Cathy Eng, Eduardo Vilar Sanchez, Maria Morelli, Zhiqin Jiang, Rajyalakshmi Luthra, Dipen M. Maru, Funda Meric-Bernstam i Scott Kopetz. "Clinicopathologic features of KRAS-mutated colorectal tumors vary by site of mutation." Journal of Clinical Oncology 31, nr 15_suppl (20.05.2013): 3632. http://dx.doi.org/10.1200/jco.2013.31.15_suppl.3632.
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3632 Background: KRAS mutations in codons 12 and 13 are present in approximately 40% of all colon cancers and predict a lack of response to monoclonal antibodies to the EGFR among patients with metastatic disease. Activating mutations in codons 61 and 146 occur less frequently, and the clinicopathologic features of this subpopulation of KRAS-mutant colorectal tumors have not been clearly defined. Methods: Records of patients treated at MD Anderson Cancer Center between December 2000 and August 2012 for metastatic colorectal cancer whose tumors underwent testing for a KRAS mutation were reviewed for clinical characteristics, concurrent mutations, and survival outcomes. Associations between mutation status and clinic features were measured by calculation of odds ratios, and survival was estimated according to the Kaplan-Meier method. Results: Among the 487 records reviewed, 225 KRAS 12/13 mutations (47.2%) and 14 KRAS 61/146 mutations (2.9%) were detected. Liver metastases were less common for patients with KRAS 61/146 mutations than for patients with KRAS 12/13 mutations (OR 0.26, p-value=0.02). No difference in lung metastases was identified for KRAS 61/146 mutated patients when compared to those with KRAS wild-type tumors (OR=2.1, p-value=0.26), even though lung metastases were more common for patients with KRAS 12/13 mutations (OR 2.86, p-value=0.001). There was no association between the presence of a KRAS 61/146 mutation and gender, stage at presentation, site of primary tumor, body mass index, tobacco history, or concurrent PIK3CA mutation. Whereas a worse survival difference from the time of metastasis detection was noted for KRAS 12/13 patients when compared to those with KRAS wild-type tumors (HR 1.74, p-value= 0.002), patients with KRAS 61/146 mutations demonstrated no change in survival outcome (HR 1.10, p-value=0.87). Conclusions: Patients with KRAS 61/146 mutations have different patterns of metastases compared to KRAS 12/13, but do not appear to share the poor prognosis associated with the more common KRAS 12/13. These results suggest that clinical phenotypes for patients afflicted with colorectal cancer may differ based on the specific codon mutated within the KRAS oncogene.
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Davis, Brad H., Art F. Y. Poon i Michael C. Whitlock. "Compensatory mutations are repeatable and clustered within proteins". Proceedings of the Royal Society B: Biological Sciences 276, nr 1663 (25.02.2009): 1823–27. http://dx.doi.org/10.1098/rspb.2008.1846.
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Compensatory mutations improve fitness in genotypes that contain deleterious mutations but have no beneficial effects otherwise. As such, compensatory mutations represent a very specific form of epistasis. We show that intragenic compensatory mutations occur non-randomly over gene sequence. Compensatory mutations are more likely to appear at some sites than others. Moreover, the sites of compensatory mutations are more likely than expected by chance to be near the site of the original deleterious mutation. Furthermore, compensatory mutations tend to occur more commonly in certain regions of the protein even when controlling for clustering around the site of the deleterious mutation. These results suggest that compensatory evolution at the protein level is partially predictable and may be convergent.
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Ramadhan, Dwi Syah Fitra, i Daryono H. Tjahjono. "Prediksi dan Identifikasi Struktur Protein EGFR Kanker Paru dengan Mutasi Titik L718Q/T790M Secara Pemodelan Homologi In Silico". Jurnal Sains dan Kesehatan 2, nr 4 (31.12.2020): 491–96. http://dx.doi.org/10.25026/jsk.v2i4.257.
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EGFR receptors play an important role in the growth of cancer cells, and these receptors have undergone various types of mutations. At this time, the effect of the L718Q / T790M point mutation on the EGFR receptor is not known, therefore the aim of this study is to predict the EGFR structure with the L718Q / T790M point mutation using in silico homology modeling. The mutant protein was successfully modeled using SWISS-Model expasy webserver and showed good evaluation results after the protein was minimized as indicated by the results of the Ramachandran outlier score of 0%, clashscore 0.98, and MolProbity 1.15. Identification of the active site of the mutant protein shows a conformational change of the active site that causes a steric collision between the inhibitor group and the amino acid side chain of the mutant protein.
Keywords: EGFR, mutation, homology modeling, in silico.
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Freije, José M. P., Pilar Blay, Nicholas J. MacDonald, Richard E. Manrow i Patricia S. Steeg. "Site-directed Mutation of Nm23-H1". Journal of Biological Chemistry 272, nr 9 (28.02.1997): 5525–32. http://dx.doi.org/10.1074/jbc.272.9.5525.
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TAKAGI, Hitoshi, i Masatomo MORI. "New Mutation Site of Cholinesterase Gene". Internal Medicine 36, nr 1 (1997): 1–2. http://dx.doi.org/10.2169/internalmedicine.36.1.
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Yun, Jiwon, Jung-Ah Kim, Byungjin Hwang, Hee Sue Park, Kyongok Im, Sung-Min Kim, Dajeong Jeong, Kyu Min Lim, Duhee Bang i Dong Soon Lee. "Triple-Negative Myeloproliferative Neoplasms Vs. Calr, JAK2 or MPL-Mutated Myeloproliferative Neoplasms: Distinct Molecular Characteristics". Blood 132, Supplement 1 (29.11.2018): 1772. http://dx.doi.org/10.1182/blood-2018-99-118013.
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Abstract Background: We compared the clinical, cytogenetic, molecular features, and telomere lengths of patients with triple negative MPN and MPN with any of CALR, JAK2 or MPL mutations. Methods: Target capture sequencing of 87 genes and molecular cytogenetic studies were performed in 61 Korean patients with MPN. Also, we searched the newly reported mutations and novel mutations in triple negative MPN [JAK2-G335D (germline), JAK2-F556V, JAK2-G571S (germline), JAK2-V625F (germline), MPL-T119I, MPL-S204F/P, MPL-E230G, MPL-V285E (germline), MPL-R321W (germline), MPL-Y591D, MPL-S505N and MPL-W515R]. We compared clinical and molecular characteristics between two groups. Additionally, we performed telomere quantitative FISH for 48 patients' samples and measured telomere/centromere ratios of them. Results: Among 61 patients, 13 patients showed mutations in CALR, 34 in JAK2, and 3 in MPL. All of JAK2 mutation and CALR mutation site were reported sites, but 2 among 5 mutation site of MPL were novel mutation [D128N, D261Y]. Twelve patients showed triple negative (7 of PMF 7, 2 of ET, and 3 of MPN-U) - they showed 8 different mutation sites among 6 different genes (ASXL1, DNMT3A, NPM1, POLG, SRSF2, and ZMYM3). NPM1, POLG, and ZMYM3 mutations were seen only in triple negative patients. NPM1 mutation was significantly higher in triple negative MPN (P=0.0301). In telomere length study, there was no statistical difference between triple negative group (T/C ratio mean 12.5) and CALR, JAK2 or MPL mutated group (T/C ratio mean 10.0). Although, MPN patients with telomere length shorter than normal control's lower 10% (7.04) showed poor prognosis (P=0.0045). Conclusions: Patients with triple negative MPN are characterized by high frequency of NPM1 mutation and less number of somatic mutations. Since the mutational analysis for diagnostic purposes is limited to exons 14 of JAK2, exon 10 of MPL and exon 9 of CALR at present, search for JAK2 and MPL mutations in other sites are essential in triple negative MPNs. Keywords: Myeloproliferative neoplasms, next-generation sequencing, triple negative MPN, chromosome, FISH, telomere Disclosures No relevant conflicts of interest to declare.
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Hall, Michael J., Michelle J. McSweeny, Kim Rainey, Hannah Campbell, Chau Nguyen i Catherine Neumann. "Risks and implications of multiple actionable pathogenic germline variants discovered by panel-based cancer predisposition testing." Journal of Clinical Oncology 41, nr 4_suppl (1.02.2023): 792. http://dx.doi.org/10.1200/jco.2023.41.4_suppl.792.
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792 Background: Multi-gene hereditary cancer panels have revolutionized how patients with germline mutations are identified by testing multiple genes at the same time. Despite the availability of panel testing, many patients with a known familial mutation will only undergo single site genetic testing due to limitations in guideline recommendations and insurance coverage. This approach risks a failure to detect additional pathogenic variants and an inappropriate management of cancer risk. In our clinical experience, a subset of patients pursue multigene testing despite a known familial mutation. Our group has identified patients who carry more than one mutation and mutations that would have been missed if the patients had only undergone single site testing. We investigated the patients and families from our risk assessment clinic with multiple familial mutations and determined how medical management may have been changed due to the presence of multiple mutations in family. Methods: The Fox Chase Cancer Center Risk Assessment Program (RAP) Registry was queried to identify patients who carry more than one mutation. Pedigrees of patients and families identified with multiple germline mutations were reviewed. Screening management guidelines were determined from the most recent NCCN guidelines published at the time the patient tested (Genetic/Familial High-Risk Assessment: Breast, Ovarian, and Pancreatic and Genetic/Familial High-Risk Assessment: Colorectal). The RAP Registry is an IRB approved protocol (IRB 09-831). Results: 70 patients were found to carry at least 2 mutations (excluding patients with biallelic MUTYH mutations) since introducing multi-gene panel testing in 2014. The most common second mutation was the I1307K variant in the APC gene at 20% (14/70). We also identified 20 patients who would have received incomplete genetic risk assessment if they only underwent single site testing and screening management changed in 60% (12/20) of these patients. 35% (7/20) of these patients did not meet NCCN criteria for additional germline testing beyond single site testing. Conclusions: Multi-gene hereditary cancer panels identify patients and families with multiple germline mutations. Patients undergoing single site cascade testing are at risk of receiving inaccurate risk assessment based on incomplete ascertainment of germline cancer risks. Detection of additional actionable mutations will frequently lead to changes in medical management.
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Zhou, Rong-Fu, Zhou Na i OuYang Jian. "Studies on the Genetic Mutations of Hereditary Fibrinogen Disorder". Blood 128, nr 22 (2.12.2016): 4954. http://dx.doi.org/10.1182/blood.v128.22.4954.4954.
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Abstract Hereditary fibrinogen disorder is a rare kind of bleeding disease, which divided into two types. Type I is a kind of quantity disorder, including afibrinogenemia and hypofibrinogenemia. Type II is a kind of quality disorder, including dysfibrinogenemia. Fibrinogen is a kind of hexameric glycoprotein and consists of two pairs of three chains, which are Aα, Bβ and γchain. FGA、FGB and FGG code for the relevant glycoprotein. The mutations on these genes are responsible for this disorder. In this study, the levels of fibrinogen antigen of 12 cases with low fibrinogen activity were firstly detected. The results showed that one case had low level, and the patient definded as hypofibrinogenemia, and other 11 cases had normal level, thus these patients identified as dysfibrinogenemia. All exons and their flanks of FGA , FGB and FGG were amplified by PCR. THe PCR products were sequenced directly and blasted to normal sequence of corresponding gene to find the mutation. The result showed that among 11 cases with dysfibrinogenemia, five harbored Aα Arg16His heterozygous mutation and one of those also possessed a de novo γAsp185Asn heterozygous mutation ; one Aα Arg16Cys heterozygous mutation; four γArg275Cys heterozygous mutation and one γArg275His heterozygous mutation.The patient with hypofibrinogenemia harbored Aα Cys36Arg heterozygous mutation. Endonuclease restriction digestion was performed to exclude genetic polymorphism for the γAsp185Asn mutation. In molecular modeling, the hydrogen bonds were changed in the mutational variant of γ185Asn. Besides, a new site of glycosylation might appear after the mutation, which might lead to destroy the stability of molecular structure. The alignment of homologous sequence between different species suggested that γAsp185 was a highly conservative site. In a word, the above mutations might be the causes of dysfibrinogenemia or hypofibrinogenemia for these patients. Disclosures No relevant conflicts of interest to declare.
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Koenig, Patrick, Chingwei V. Lee, Benjamin T. Walters, Vasantharajan Janakiraman, Jeremy Stinson, Thomas W. Patapoff i Germaine Fuh. "Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding". Proceedings of the National Academy of Sciences 114, nr 4 (5.01.2017): E486—E495. http://dx.doi.org/10.1073/pnas.1613231114.
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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.
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Askew, G. R., T. Doetschman i J. B. Lingrel. "Site-directed point mutations in embryonic stem cells: a gene-targeting tag-and-exchange strategy". Molecular and Cellular Biology 13, nr 7 (lipiec 1993): 4115–24. http://dx.doi.org/10.1128/mcb.13.7.4115-4124.1993.
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Sequential gene targeting was used to introduce point mutations into one alpha 2 isoform Na,K-ATPase homolog in mouse embryonic stem (ES) cells. In the first round of targeted replacement, the gene was tagged with selectable markers by insertion of a Neor/HSV-tk gene cassette, and this event was selected for by gain of neomycin (G418) resistance. In the second targeted replacement event, the tagged genomic sequence was exchanged with a vector consisting of homologous genomic sequences carrying five site-directed nucleotide substitutions. Embryonic stem cell clones modified by exchange with the mutation vector were selected for loss of the HSV-tk gene by resistance to ganciclovir. Candidate clones were further screened and identified by polymerase chain reaction and Southern blot analysis. By this strategy, the endogenous alpha 2 isoform Na,K-ATPase gene was altered to encode two other amino acids so that the enzyme is resistant to inhibition by cardiac glycosides while maintaining its transmembrane ion-pumping function. Since the initial tagging event and the subsequent mutation-exchange event are independent of one another, a tagged cell line can be used to generate a variety of mutant lines by exchange with various mutation vectors at the tagged locus. This method should be useful for testing specific mutations introduced into the genomes of tissue culture cells and animals and for developing animal models encompassing the mutational variability of known genetic disorders.
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Askew, G. R., T. Doetschman i J. B. Lingrel. "Site-directed point mutations in embryonic stem cells: a gene-targeting tag-and-exchange strategy." Molecular and Cellular Biology 13, nr 7 (lipiec 1993): 4115–24. http://dx.doi.org/10.1128/mcb.13.7.4115.
Pełny tekst źródłaStreszczenie:
Sequential gene targeting was used to introduce point mutations into one alpha 2 isoform Na,K-ATPase homolog in mouse embryonic stem (ES) cells. In the first round of targeted replacement, the gene was tagged with selectable markers by insertion of a Neor/HSV-tk gene cassette, and this event was selected for by gain of neomycin (G418) resistance. In the second targeted replacement event, the tagged genomic sequence was exchanged with a vector consisting of homologous genomic sequences carrying five site-directed nucleotide substitutions. Embryonic stem cell clones modified by exchange with the mutation vector were selected for loss of the HSV-tk gene by resistance to ganciclovir. Candidate clones were further screened and identified by polymerase chain reaction and Southern blot analysis. By this strategy, the endogenous alpha 2 isoform Na,K-ATPase gene was altered to encode two other amino acids so that the enzyme is resistant to inhibition by cardiac glycosides while maintaining its transmembrane ion-pumping function. Since the initial tagging event and the subsequent mutation-exchange event are independent of one another, a tagged cell line can be used to generate a variety of mutant lines by exchange with various mutation vectors at the tagged locus. This method should be useful for testing specific mutations introduced into the genomes of tissue culture cells and animals and for developing animal models encompassing the mutational variability of known genetic disorders.
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Chang, JG, PH Chen, SS Chiou, LS Lee, LI Perng i TC Liu. "Rapid diagnosis of beta-thalassemia mutations in Chinese by naturally and amplified created restriction sites". Blood 80, nr 8 (15.10.1992): 2092–96. http://dx.doi.org/10.1182/blood.v80.8.2092.2092.
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Abstract We developed a rapid and simple method to diagnose the molecular defects of beta-thalassemia in Chinese patients. This method involves the selective amplification of a DNA fragment from human beta globin gene with specific oligonucleotide primers, followed by digestion with restriction enzymes that recognize artificially created or naturally occurring restriction sites. To detect the 4-nucleotide deletion of codon 41–42, we introduced a single mismatch nucleotide into the 3′ end of the upstream primer to create an artificial Taq I restriction site. With a similar approach, an artificial Rsa I site was generated to detect the nucleotide 654 mutation (C-->T) of IVS-2, and Alu I restriction site was created to detect the codon 17 mutation (A-->T), and EcoRI restriction site was created for the -28 mutation (A-->G), a Rsa I restriction site was created for the nucleotide 5 mutation (G-- >C) of IVS-1, and a Spe I restriction site was created to distinguish the codon 71 (+T) and codon 71/72 (+A) mutations from a normal sequence. The other eight rare mutations that occur in the genes of the Chinese people naturally create or abolish restriction sites. Using this kind of approach, we are able to provide a simple, rapid, accurate, and nonradioactive method to detect the genetic defects of beta-thalassemia in the Chinese population. It should be used not only for routine screening but also for prenatal diagnosis.
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Chang, JG, PH Chen, SS Chiou, LS Lee, LI Perng i TC Liu. "Rapid diagnosis of beta-thalassemia mutations in Chinese by naturally and amplified created restriction sites". Blood 80, nr 8 (15.10.1992): 2092–96. http://dx.doi.org/10.1182/blood.v80.8.2092.bloodjournal8082092.
Pełny tekst źródłaStreszczenie:
We developed a rapid and simple method to diagnose the molecular defects of beta-thalassemia in Chinese patients. This method involves the selective amplification of a DNA fragment from human beta globin gene with specific oligonucleotide primers, followed by digestion with restriction enzymes that recognize artificially created or naturally occurring restriction sites. To detect the 4-nucleotide deletion of codon 41–42, we introduced a single mismatch nucleotide into the 3′ end of the upstream primer to create an artificial Taq I restriction site. With a similar approach, an artificial Rsa I site was generated to detect the nucleotide 654 mutation (C-->T) of IVS-2, and Alu I restriction site was created to detect the codon 17 mutation (A-->T), and EcoRI restriction site was created for the -28 mutation (A-->G), a Rsa I restriction site was created for the nucleotide 5 mutation (G-- >C) of IVS-1, and a Spe I restriction site was created to distinguish the codon 71 (+T) and codon 71/72 (+A) mutations from a normal sequence. The other eight rare mutations that occur in the genes of the Chinese people naturally create or abolish restriction sites. Using this kind of approach, we are able to provide a simple, rapid, accurate, and nonradioactive method to detect the genetic defects of beta-thalassemia in the Chinese population. It should be used not only for routine screening but also for prenatal diagnosis.
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Misawa, Kazuharu, i Fumio Tajima. "Estimation of the Amount of DNA Polymorphism When the Neutral Mutation Rate Varies Among Sites". Genetics 147, nr 4 (1.12.1997): 1959–64. http://dx.doi.org/10.1093/genetics/147.4.1959.
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Abstract Knowing the amount of DNA polymorphism is essential to understand the mechanism of maintaining DNA polymorphism in a natural population. The amount of DNA polymorphism can be measured by the average number of nucleotide differences per site (π), the proportion of segregating (polymorphic) site (s) and the minimum number of mutations per site (s*). Since the latter two quantities depend on the sample size, θ is often used as a measure of the amount of DNA polymorphism, where θ = 4Nμ, N is the effective population size and μ is the neutral mutation rate per site per generation. It is known that θ estimated from π, s and s* under the infinite site model can be biased when the mutation rate varies among sites. We have therefore developed new methods for estimating θ under the finite site model. Using computer simulations, it has been shown that the new methods give almost unbiased estimates even when the mutation rate varies among sites substantially. Furthermore, we have also developed new statistics for testing neutrality by modifying Tajima's D statistic. Computer simulations suggest that the new test statistics can be used even when the mutation rate varies among sites.