Academic literature on the topic 'SITE MUTATION'
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Journal articles on the topic "SITE MUTATION"
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Ozdemir, D., P. S. Hart, O. H. Ryu, S. J. Choi, M. Ozdemir-Karatas, E. Firatli, N. Piesco, and T. C. Hart. "MMP20 Active-site Mutation in Hypomaturation Amelogenesis Imperfecta." Journal of Dental Research 84, no. 11 (November 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, and R. Cellerino. "MSH2 splice site mutation and endometrial cancer." International Journal of Gynecologic Cancer 16, no. 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, and Max L. Nibert. "Thermolabilizing Pseudoreversions in Reovirus Outer-Capsid Protein μ1 Rescue the Entry Defect Conferred by a Thermostabilizing Mutation." Journal of Virology 81, no. 14 (May 16, 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, and R. A. Weisberg. "The effect of attachment site mutations on strand exchange in bacteriophage lambda site-specific recombination." Genetics 122, no. 4 (August 1, 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, and Michael Sangmin Lee. "Characterization of the number and site of APC mutations in sporadic colorectal cancer." Journal of Clinical Oncology 35, no. 4_suppl (February 1, 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, and Hidehiko Saito. "Two Distinct Novel Splice Site Mutations in a Compound Heterozygous Patient with Protein S Deficiency." Thrombosis and Haemostasis 77, no. 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, and 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, no. 22 (September 9, 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, and 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, no. 9 (September 1, 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, and 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, no. 8 (August 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, and S. R. Wayne Chen. "The central domain of cardiac ryanodine receptor governs channel activation, regulation, and stability." Journal of Biological Chemistry 295, no. 46 (September 2, 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.
Dissertations / Theses on the topic "SITE MUTATION"
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Patel, Premal Harshad. "Evolution of DNA polymerase active site /." Thesis, Connect to this title online; UW restricted, 2001. http://hdl.handle.net/1773/6361.
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Marcello, Matthew R. "Analysis of recombinant human prostasin carrying a serine active site mutation." Honors in the Major Thesis, University of Central Florida, 2003. http://digital.library.ucf.edu/cdm/ref/collection/ETH/id/325.
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This item is only available in print in the UCF Libraries. If this is your Honors Thesis, you can help us make it available online for use by researchers around the world by following the instructions on the distribution consent form at http://library.ucf.edu/Systems/DigitalInitiatives/DigitalCollections/InternetDistributionConsentAgreementForm.pdf You may also contact the project coordinator, Kerri Bottorff, at kerri.bottorff@ucf.edu for more information.Bachelors
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Molecular Biology and Microbiology
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Wenwieser, Sandra Verena Corinna Tina. "Subunit interactions in regulation and catalysis of site-specific recombination." Thesis, University of Glasgow, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.343974.
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Chitpinityol, Supannee. "Heterologous expression and site-directed mutagenesis of the enzyme chymosin." Thesis, University of Reading, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.320101.
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Sheikh, Qaiser Iftikhar. "Exploring the structure and function of bacterial cytosine specific DNA methyltransferases using site-directed mutagenesis." Thesis, University of Sheffield, 2001. http://etheses.whiterose.ac.uk/10258/.
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Point mutations were engineered into the sequence of the multispecific DNA methyltransferase (Mtase) M. SPRI in motif IX, in order to mimic the corresponding motif IX of mono-specific Mtase. A similar approach was adopted to modify the sequence of the monospecific enzyme M. HhaI in motifs IX and X based on the available structure and as a consequence the enzyme regained methylation potential. It was thought that these changes might be sufficient to enable functional exchange of the target recognition domains (TRDs) between a mono- and a multispecific enzyme. However, insertion of various segments of TRD region from M. SPRI into the M. HhaI was not successful (Chapter 4). To establish whether mono- and multispecific Mtases are incompatible in terms of sequence exchanges, a systematic "swapping" of motifs was carried out (Chapter 5). These experiments suggested that there are some enzyme-specific structural interactions between different subunits within each class of Mtases. In second half of this thesis a bacterial two-hybrid system based on the reversible assembly of an engineered form of M. SPRI was developed (Chapter 6). However the Mtase protein does not assemble into an active species until a DNA segment encoding a leucine zipper motif is fused to each of the two halves. Co-transformation of E. coli with the plasmids expressing the C-terminal and N-terminal domains respectively resulted in the abolition of colonies on double antibiotic plates, when an mcr strain was used as host. High performance liquid chromatography was used to estimate the extent of modification of plasmids indirectly. The extent of methylation at specific sequences within a plasmid molecule was readily detected by the corresponding differential susceptibility to digestion by specific restriction enzymes. Using this approach it proved possible to detect different levels of activity produced by wild type and mutant recombinant DNA Methyltransferases with sensitivity and in a semi quantitative manner. In order to analyse the biochemical properties of Mtase, I have developed an in vitro translation-modification assay. Binary studies with the mutants (from Chapter 3 and 5) showed that there were no detectable sequence-specific recognition differences between these enzymes. Taken together, these results suggest that motif IX plays a role in general stabilisation of the enzyme core structure and has a less significant role in DNA recognition.
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Dinda, Stephen B. "Predicting RNA Mutation Using 3D Structure." Bowling Green State University / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1321280932.
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Tinteroff, Gil Vanessa. "De Paracas à Nasca sur la côte du sud du Pérou : archéologie d'une mutation culturelle." Paris 4, 2008. http://www.theses.fr/2008PA040039.
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Sur la côte sud du Pérou, à l'issu du déclin de la civilisation Chavín, la culture Paracas laisse peu à peu place à la culture Nasca. Datée entre 200 avant J. -C. Et 100 de notre ère, cette période, communément appelée "transition Paracas – Nasca" sur la côte sud, est celle de nombreux changements culturels. À travers l’étude de divers contextes archéologiques de cette région, et en particulier de Necrópolis dans la péninsule de Paracas et de Cahuachi dans la vallée de Nasca, cette thèse se propose de définir les causes, les mécanismes socioculturels et les acteurs culturels des transformations qui s'opèrent sur la côte sud au cours de cette période charnière de l'histoire du Pérou préhispanique. Elle offre une définition des cultures Paracas, Topará et Nasca, de leurs origines, de leur développement et de leurs liens culturels, géographiques et chronologiquesOn the south coast of the Peru, after the decline of the Chavin civilisation, Paracas culture is slowly making way for Nasca culture. Dated between 200 BC and 100 AD, this period, commonly called "the Paracas – Nasca transition" on the south coast, is the one of numerous cultural changes. Through the study of different archaeological contexts of this region, particularly of Necrópolis in the Paracas peninsula and of Cahuachi in the Nasca valley, the purpose of this thesis is to define the causes, the sociocultural mechanisms and the cultural players of the changes occuring in the south coast during this turning point in the prehispanic Peru history. This thesis offers a definition of Paracas, Topará and Nasca cultures, of their origins, their development and their cultural, geographical and chronogical relations
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Lefebvre, Anne. "Le site CpG dans l'ADN : impact possible des variations conformationnelles sur le taux de mutations." Châtenay-Malabry, Ecole centrale de Paris, 1996. http://www.theses.fr/1996ECAP0485.
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Nous avons analysé la structure du dinucléotide CpG en fonction de la séquence d'ADN qui le contient, par résonance magnétique nucléaire et modélisation moléculaire, pour déterminer en quoi la structure de CpG influe sur les modifications structurales induites par la méthylation de la cytosine de ce dinucléotide, et mettre en relation la structure de CpG et le taux de mutations observé sur ces sites. La permutation de ses plus proches voisins modifie fortement la conformation de CpG. Au sein de la tétrade ACGT, comme dans d(GTACGTAC)2, il adopte un grand twist, associé à une phase élevée de la guanine. Au sein de d(CATCGATG)2, la structure de CpG est beaucoup plus malléable, et il adopte des valeurs moyennes de twist et de phase de la guanine plus faibles. Mais, la conformation de CpG est également influencée par des résidus plus éloignés dans la séquence, puisque la structure qu'il adopte dans d(CTTCGAAG)2 ressemble plus à celle calculée pour d(GTACGTAC)2 que pour d(CATCGATG)2. Nous avons également montré que les effets structuraux de la méthylation de CpG dépendent nettement de la conformation initiale de ce dinucléotide : la méthylation de la cytosine centrale modifie la structure moyenne de d(CATCGATG)2 mais pas de d(CTTCGAAG)2. Enfin, dans ces deux derniers oligonucléotides, un équilibre BI/BII a été mis en évidence au niveau du squelette de CpG, la proportion de conformères BII étant plus importante dans d(CTTCGAAG)2. Remarquablement, le double mésappariement d(TpG)2 dans d(CTTTGAAG)2 adopte un équilibre BI/BII très similaire à celui observé pour CpG dans d(CTTCGAAG)2. La structure particulière de CpG dans certains contextes d'ADN, en l'absence de méthylation, suffirait donc à expliquer sa reconnaissance par des enzymes de réparation, et donc sa mutation.
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Sada, Yoshinao. "Genetic studies on the target-site resistance to sulfonylurea herbicides in Schoenoplectus juncoides." Kyoto University, 2014. http://hdl.handle.net/2433/193553.
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Liu, Fengling. "Kinetic and Crystallographic Studies of Drug-Resistant Mutants of HIV-1 Protease: Insights into the Drug Resistance Mechanisms." Digital Archive @ GSU, 2007. http://digitalarchive.gsu.edu/biology_diss/19.
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HIV-1 protease (PR) inhibitors (PIs) are important anti-HIV drugs for the treatment of AIDS and have shown great success in reducing mortality and prolonging the life of HIV-infected individuals. However, the rapid development of drug resistance is one of the major factors causing the reduced effectiveness of PIs. Consequently, various drug resistant mutants of HIV-1 PR have been extensively studied to gain insight into the mechanisms of drug resistance. In this study, the crystal structures, dimer stabilities, and kinetics data have been analyzed for wild type PR and over 10 resistant mutants including PRL24I, PRI32V, PRM46L, PRG48V, PRI50V, PRF53L, PRI54V, PRI54M, PRG73S and PRL90M. These mutations lie in varied structural regions of PR: adjacent to the active site, in the inhibitor binding site, the flap or at protein surface. The enzymatic activity and inhibition were altered in mutant PR to various degrees. Crystal structures of the mutants complexed with a substrate analog inhibitor or drugs indinavir, saquinavir and darunavir were determined at resolutions of 0.84 – 1.50 Å. Each mutant revealed distinct structural changes, which are usually located at the mutated residue, the flap and inhibitor binding sites. Moreover, darunavir was shown to bind to PR at a new site on the flap surface in PRI32V and PRM46L. The existence of this additional inhibitor binding site may explain the high effectiveness of darunavir on drug resistant mutants. Moreover, the unliganded structure PRF53L had a wider separation at the tips of the flaps than in unliganded wild type PR. The absence of flap interactions in PRF53L suggests a novel mechanism for drug resistance. Therefore, this study enhanced our understanding of the role of individual residues in the development of drug resistance and the structural basis of drug resistance mechanisms. Atomic resolution crystal structures are valuable for the design of more potent protease inhibitors to overcome the drug resistance problem.
Books on the topic "SITE MUTATION"
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Institut d'aménagement et d'urbanisme de la région d'Île de France., ed. La mutation du site de Billancourt: Contexte et enjeu d'un projet. Paris: I.A.U.R.I.F., 1990.
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McPherson, M.J., (Ed.), ed. Directed Mutagenesis: A Practical Approach. Oxford: I.R.L. P., 1991.
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J, McPherson M., ed. Directed mutagenesis: A practical approach. Oxford [England]: IRL Press, 1991.
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R, Ballain, ed. Sites urbains en mutation: Territoires et trajectoires. Paris: L'Harmattan, 1990.
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Meslé, Jean-Yves. SMN, Société métallurgique de Normandie: Mémoires et mutations d'un site industriel. Bayeux: OREP, 2013.
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Khan, Amir R. Mutational and structural analysis of second-site transmembrane region mutants of phage M13 coat protein. Ottawa: National Library of Canada, 1993.
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McPherson, M. J. Directed Mutagenesis: A Practical Approach (Practical Approach Series). Oxford University Press, USA, 1991.
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McPherson, M. J. Directed Mutagenesis: A Practical Approach (The Practical Approach Series). Oxford University Press, USA, 1991.
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Bergmann, Carsten, and Klaus Zerres. Autosomal recessive polycystic kidney disease. Edited by Neil Turner. Oxford University Press, 2015. http://dx.doi.org/10.1093/med/9780199592548.003.0313.
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Autosomal recessive polycystic kidney disease (ARPKD) is an important cause of childhood renal- and liver-related morbidity and mortality with variable disease expression. Many patients manifest peri- or neonatally with a mortality rate of 30–50%, whereas others survive to adulthood with only minor clinical features. ARPKD is typically caused by mutations in the PKHD1 gene that encodes a 4074-amino acid type 1 single-pass transmembrane protein called fibrocystin or polyductin. Fibrocystin/polyductin is among other cystoproteins expressed in primary cilia, basal bodies, and centrosomes, but its exact function has still not been fully unravelled. Mutations were found to be scattered throughout the gene with many of them being private to single families. Correlations have been drawn for the type of mutation rather than for the site of the individual mutation. Virtually all patients carrying two truncating mutations display a severe phenotype with peri- or neonatal demise while surviving patients bear at least one hypomorphic missense mutation. However, about 20–30% of all sibships exhibit major intrafamilial phenotypic variability and it becomes increasingly obvious that ARPKD is clinically and genetically much more heterogeneous and complex than previously thought.
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Walsh, Bruce, and Michael Lynch. The Genetic Effective Size of a Population. Oxford University Press, 2018. http://dx.doi.org/10.1093/oso/9780198830870.003.0003.
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The effects of genetic drift usually assume an idealized population of constant size. This chapter shows how the population size for such an idealized population can be replaced with an effective population size for populations with age structure, unequal sex ratios, a history of expansion or contraction, inbreeding, and population subdivision. These demographic features impact the entire genome more or less equally. A relatively recent understanding is that selection at a site can dramatically reduce the local effective population size experienced by nearby linked sites (the Hill-Robertson effect). This can arise from background selection to remove deleterious new mutations or from selective sweeps wherein favorable new mutations are driven toward fixation. The Hill-Robertson effect is a general way to describe the fact that selection at a site makes selection are other linked sites less efficient, and, therefore, more neutral. This chapter discusses the implications of this finding for genome structure.
Book chapters on the topic "SITE MUTATION"
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Gurushidze, Maia, Stefan Hiekel, Ingrid Otto, Götz Hensel, and Jochen Kumlehn. "Site-Directed Mutagenesis in Barley by Expression of TALE Nuclease in Embryogenic Pollen." In Biotechnologies for Plant Mutation Breeding, 113–28. Cham: Springer International Publishing, 2016. http://dx.doi.org/10.1007/978-3-319-45021-6_7.
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Polgár, L., and M. L. Bender. "Simulated Mutation at the Active Site of Biologically Active Proteins." In Advances in Enzymology - and Related Areas of Molecular Biology, 381–400. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2006. http://dx.doi.org/10.1002/9780470122785.ch8.
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Remya, S., and K. Praveen. "Protecting the Augmented Browser Extension from Mutation Cross-Site Scripting." In Advances in Intelligent Systems and Computing, 215–23. New Delhi: Springer India, 2015. http://dx.doi.org/10.1007/978-81-322-2517-1_22.
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Cohen, Y. Haimi, R. Bargal, M. Zeigler, T. Markus-Eidlitz, V. Zuri, and A. Zeharia. "Hyperargininemia: A Family with a Novel Mutation in an Unexpected Site." In JIMD Reports, 83–88. Berlin, Heidelberg: Springer Berlin Heidelberg, 2011. http://dx.doi.org/10.1007/8904_2011_101.
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Maguire, D. J., A. J. Sun, and W. Rosner. "A Possible New mtrRNA Mutation Site for Aminoglycoside-Induced Deafness Syndrome." In Advances in Experimental Medicine and Biology, 411–17. Boston, MA: Springer US, 1999. http://dx.doi.org/10.1007/978-1-4615-4717-4_50.
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Temme, Manfred. "Somatic Mutation in the Polynesian Rat (Rattus Exulans) at Enewetak Nuclear Test Site." In Current Mammalogy, 483–93. Boston, MA: Springer US, 1987. http://dx.doi.org/10.1007/978-1-4757-9909-5_12.
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Kagamiyama, H., S. Kuramitsu, Y. Inoue, S. Tanase, and Y. Morino. "Mutation of Lysine 258 to Arginine in the Active Site of Aspartate Aminotransferase." In Biochemistry of Vitamin B6, 69–72. Basel: Birkhäuser Basel, 1987. http://dx.doi.org/10.1007/978-3-0348-9308-4_13.
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Donovan, Kathleen A., Sujay K. Singh, and Chella S. David. "Mutation in the Aβ Gene of B6.C-H-2bm12. Antibody Binding Site VS. T Cell Recognition Sites." In H-2 Antigens, 315–20. Boston, MA: Springer US, 1987. http://dx.doi.org/10.1007/978-1-4757-0764-9_31.
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Rignall, Tauna R., John O. Baker, Suzanne L. McCarter, William S. Adney, Todd B. Vinzant, Stephen R. Decker, and Michael E. Himmel. "Effect of Single Active-Site Cleft Mutation on Product Specificity in a Thermostable Bacterial Cellulase." In Biotechnology for Fuels and Chemicals, 383–94. Totowa, NJ: Humana Press, 2002. http://dx.doi.org/10.1007/978-1-4612-0119-9_32.
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Nakagawa, Hitoshi. "History of mutation breeding and molecular research using induced mutations in Japan." In Mutation breeding, genetic diversity and crop adaptation to climate change, 24–39. Wallingford: CABI, 2021. http://dx.doi.org/10.1079/9781789249095.0003.
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Abstract Following the construction of the Gamma Field at the Institute of Radiation Breeding in 1960, mutation breeding was accelerated in Japan. The facility is used, with a radiation dose up to 2 Gy/day (ca. 300,000 times that of natural background), to induce mutations at a higher frequency than occurs in nature. There have been 318 direct- use mutant cultivars representing 79 species generated through irradiation of gamma-rays, X-rays, ion beams and chemicals and somaclonal variation. Approximately 79% of these direct-use cultivars were induced by radiation. There have been 375 indirect-use mutant cultivars, including 332 rice, of which 162 cultivars (48.8%) were derived from the semi-dwarf mutant cv. 'Reimei'. The economic impact of these mutant cultivars, primarily of rice and soybean, is very large. Some useful mutations are discussed for rice, such as low digestible protein content, low amylose content, giant embryo and non-shattering. Useful mutations in soybean such as radiosensitivity, fatty acid composition and super-nodulation have been identified. Japanese pear and apple resistant to Alternaria disease have also been identified. The achievements of biological research such as characterization and determination of deletion size generated by gamma-rays, the effect of deletion size and the location, and a mechanism of dominant mutation induction are identified. Similarly, genetic studies on mutations generated through the use of gamma-ray induced mutations, such as phytochrome response, aluminium tolerance, stay-green (Mendel's gene) and epicuticular wax have also been conducted. Mutation breeding is a very useful technology for isolating genes and for elucidating gene functions and metabolic pathways in various crops.
Conference papers on the topic "SITE MUTATION"
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Antonarakis, E. "The Molecular Genetics of Hemophilia A Stylianos." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643980.
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Hemophilia A is a common X linked hereditary disorder of blood coagulation due to deficiency of factor 8. The gene for factor 8 has been cloned and characterized (Nature 312:326-342, 1984). It is divided into 26 exons and 25 introns and spans 186 kb of DNA. The CGNA is 9 kb and codes for 2351 amino acids. The first 19 amino acids comprise the secretory leader peptide and the mature excreted polypeptide consists of 2332 amino acids. The nucleotide sequence of the exons and the exon-intron junctions is known and the complete amino acid sequence has been deducedSeveral laboratories have used cloned factor 8 DNA sequences as probes to characterized mutations that are responsible for hemophilia A in certain pedigrees. These mutations have been characterized by restriction analysis, oligonucleotide hybridization, cloning and sequencing of DNA from appropriate patientsIn about 500 patients with hemophilia A examined, the molecular defect has been recognized in 39. Both gross alterations (mainly deletions) and point mutations of the factor 8 gene have been found.A total of 19 different deletions have been observed. No two unrelated pedigrees share the same exact deletion.The size of the deleted DNA varies from 1.5 kb to more than 210 kb. All but one of these deletions are associated with severe hemophilia A. A deletion of 6 kb that contains exon 22 only is associated with moderate hemophilia. Some deletions are present in patients with inhibitors to factor 8. No correlation of the size or the position of the deletions can be found with the presence of inhibitors to factor 8.A total of 20 point mutations have been characterized. All are recognized by restriction analysis and involve Taq I sites. All are mutations of CpG dinucleotides and generate nonsense or missence codons. Unrelated pedigrees have the same single nucleotide change because of independent origin of the same mutation. In many instances de novo occurrence of a point mutation has been observed. CpG dinucleotides are hot spots for mutation to TG or CA presumably because of spontaneous deamination of methylcytosine. Some point mutations are present in patients with inhibitors but no correlation of the site of mutation and inhibitor formation has been found. The nonsense mutations are present in patients with severe hemophilia A. A missense mutation (Arg Gin) in exon 26 was found in a patient with mild hemophilia while another Arg Gin mutation in exon 24 has been observed in a patient with severe disease. The creation of a donor splice site in IVS 4 of factor 8 gene has been observed in a patient with mild hemophilia.Few DNA polymorphisms within the factor 8 gene and two other closely linked polymorphisms have been used for carrier detection and prenatal diagnosis of hemophilia A. These DNA markers are useful in more than 90% of families at risk for hemophilia A.The author thanks Drs. Gitschier, Din, Olek, Pirastou, Lawn for communication of their data prior to publication.The hemophilia project at Johns Hopkins was supported by an Institutional grant and NIH grant to S.S.A. and Haig H. Kazazian, Jr.
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Shahriar, Hossain, and Mohammad Zulkernine. "MUTEC: Mutation-based testing of Cross Site Scripting." In 2009 ICSE Workshop on Software Engineering for Secure Systems (SESS). IEEE, 2009. http://dx.doi.org/10.1109/iwsess.2009.5068458.
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Bernaedi, F., V. Bertagnolo, S. Bartolai, L. Rossi, F. Panicucci, and F. Conconi. "A POINT MUTATION AND A GENE DELETION OF FVIII GENE IN SEVERE HAEMOPHILIA." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644047.
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The presence of Factor VIII (FVIII) gene lesions has been investigated in 100 haemophilia A patients using cDNA probes for the 3'part of FVIII gene (exons 14-26 ).In two related severe patients without inhibitor a deletion removesthe exon 26; the gene lesion has been confirmed with several restriction enzymes and has been shown by densitometry of the autoradiographic pattern in a woman of the same family. The complete deletionof the exon 26 has been described by Gitschier et al. in a patient with inhibitor. Thus the comparison of the end points of the two deletions could help to define the mechanism originating these gene lesions and the relation between gene lesions and the presence of antibody.In a patient with severe Haemophilia and without inhibitor a mutation removing the TaqI site in the exon 24 and originating an abnormal band of 4.2 Kb has been found. A C→T transition in this TaqI site, originating a nonsense codon and a new Hindlll site, has been reported by Gitschier et al in a patient presenting inhibitor. The DNA from our patient tested with Hindlll shows a normal pattern thus indicating a C→T transition in the antisense strand. This mutation should causean aminoacid change (CGA→CAA, Arg→Gln) possiblyresponsible for the FVIII inactivation but that does not remove theantigenic determinants present in the COOH terminal part of FVIII.In addition the same mutation has been observed in an unrelated (asdemonstrated by RFLPs analysis) Italian haemophilic patient confirming the observation of Youssoufian et al that TaqI sites are mutational hot spots in FVIII gene.
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Bertagnolo, V., S. Volinia, C. Legnani, G. Rodorigo, V. De De Rosa, and F. Bernardi. "TWO FVIII GENE LESIONS DETECTED IN SEVERE AND MODERATE HAEMOPHILIA A." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644048.
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DNAs from 15 haemophilia A patients from different families have been hybridized to FVIII cDNA probes for the exons 14-26.In a severely affected patient (FVIII:C 2 %) the TaqI site of exon 24 is absent originating an abnormal band of 4.2 Kb. A C toT transition in the CG dinucleotide of the TaqI site (TCGA) is the probable gene mutation. Since the transition in the sense strand should originate an additional Hind III site, which is not detected in our patient, we infer that the mutation occurred in the antisensestrand causing an aminoacid change (CGA →CAA, Arg → Gin). This isin accordance with the low activity of FVIII and with the absence of inhibitor. Infact Gitschier et Al reported in a patient with ahigh titre of anti-FVIII antibody and with <1% FVIII activity a C → T transition in the coding strand, originating a nonsense codon in the TaqI site of exon 24.In the Hindlll pattern from a moderately affected patient (FVIII:C 4%) the fragment containing the exon 18 is 2.5 Kb in size (normal 2.6 Kb). Since the patterns with other restriction enzymes are indistinguishable from normal a small mutation originating a new Hind III site is likely. Both altered patterns have been detected in the patients' mothers.Work supported by Ricerca Sanitaria Finalizzata Regione Emilia Romagna
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Youssoufiän, H., A. Patel, D. Phillips, H. H. Kazazian, and S. E. Antonarakis. "RECURRENT MUTATIONS AND AN UNUSUAL DELETION IN HEMOPHILIA A." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644014.
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We have identified 15 mutations of the factor VIII (F8) gene from a panel of 107 patients with hemophilia A and have characterized these gene defects byrestriction analysis, oligonucleotide hybridization, cloning and DNA sequencing. Recurrent point mutations that involve CG to TG transitions were identified in exon 18, exon 22, and exon 24; a single CG to TG transition was identified in exon 23; and a CG to CA transition was identified in exon 24. In addition, a Taq I site alteration in intron 4 was identified in a patient with mild hemophilia, which arose dg. S23&in a grandpaternal germ cell. Cloning and sequencing of this region suggests the generation of a newsplice donor site. These data suggest that CG to TG transition is a prominent mechanism of mutation in hemophilia A. Six different deletions were also characterized. In one family, the deletion involved exon 26. However, the deletion endpoints in the male proband were different from those in his carrier mother, suggesting either gonadal mosaicism or a second deletion event in maternal meiosis.Of the 15 mutations, 6 occurred de novo within 2 generations: 4 in males and 2 in females. In these djg.novo mutations paternal age at conception was 35 (range = 32-38) and maternal age was 24 and 27. The ability to discover a sizable number of mutations in the F8 gene producing hemophilia A enables us to determine the frequency and nature of de novo mutations in man.
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Geddes, V. A., G. V. Louie, G. D. Brayer, and R. T. A. MacGillivray. "MOLECULAR BASIS OF HEMOPHILIA B: IDENTIFICATION OF THE DEFECT IN FACTOR IX VANCOUVER." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643872.
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Factor IX Vancouver (fIX-V) is the cause of a moderate form of hemophilia B. An individual presenting with this disorder had 2.6% of normal procoagulant activity in his plasma but had 62% of the normal factor IX antigen level. Specific antibodies showed that fIX-V contains epitopes for both the heavy and light chains of factor IXa. To identify the defect involved, DNA was isolated from the lymphocytes of the male hemophiliac. Southern blot analysis using a full-length factor IX cDNA as a hybridization probe showed no gross differences between the fIX-V gene and the normal factor IX gene. The DNA from the hemophiliac was then partially digested with Sau3A and the resulting fragments (10-20kbp in size) were ligated into the BamHI site of λEMBL3. The DNA was then packaged into phage particles in vitro, and the recombinant phage were screened with the factor IX cDNA as a probe. Eight phage were isolated that contained overlapping DNA covering the complete gene for fIX-V. DNA sequence analysis of the protein-encoding regions, the intron/exon junctions and 5'-and 3'-flanking sequences revealed a single nucleotide change from the normal factor IX gene. The codon for amino acid 397 was changed from ATA (lie) to ACA (Thr). This mutation is in the catalytic domain of factor IXa and is novel amongst those hemophilia B mutations reported to date. Based on the known three dimensional structures of the pancreatic serine proteases, trypsin, elastase and chymotrypsin, models have been constructed for the structures of the catalytic domains of both the normal and Thr-397 mutant of factor IXa. These results suggest that the Thr-397 mutation may alter the conformation of the substrate binding region in the active site of factor IXa Vancouver through the formation of a hydrogen bond between the hydroxyl group of the Thr-397 side chain and the main chain carbonyl group of Trp-385. The postulated conformational change would lead to reduced binding affinity for the factor IXa substrate resulting in a reduction in the catalytic activity of fIXa-Vancouver.Supported in part by grants from the Medical Research Council of Canada (to GDB and RTAM).
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Chini, Vasiliki, Yasser Al Sarraj, Michael Trese, Hatem El Shanti, and Marios Kambouris. "A Novel Homozygous Lrp5 Splice-site Deletion Mutation Causes Syndromic Autosomal Recessive Familial Exudative Vitreoretinopathy." In Qatar Foundation Annual Research Conference Proceedings. Hamad bin Khalifa University Press (HBKU Press), 2014. http://dx.doi.org/10.5339/qfarc.2014.hbpp0727.
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Matthews, R. J., I. R. Peake, and A. L. Bloom. "POINT-MUTATION OF FACTOR VIII CODING SEQUENCES IN HAEMOPHILIA A." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644013.
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In order to study the molecular basis of haemophilia A, DNA from 26 haemophilia A patients (8 severe with inhibitors, 13 severe noninhibitors and 5 mild/moderate) was screened by the Southern blotting method with FVIII cDNA probe A (a i.7kb Kpnl cDNA fragment that spans exons 1 to 12) probe B (a 4.7kb EcoRI cDNA fragment that contains exons 14 to 25 and part of exon 26) probe C (a 1.8kb EcoRI cDNA fragment that contains the remainder of exon 26 and probe D (an Apal/EcoRI 783bp cDNA fragment that includes all of exons 22 to 25 and parts of exons 21 and 26). All cDNA probes were kindly provided by Genetics Institutes Inc.No large structural alterations of the FVIII gene were detected in any of the patients. However altered TaqI restriction sites within the coding regions of 3 patients were observed. DNA from patient 1 with severe haemophilia (VIIIAg < 0.1 u/dl, inhibitor negative) when probed with probe C showed a substitution of the normal 2.6kb TaqI and 4.5kb EcoRI fragments with novel 12kb TaqI and 11.5kb EcoRI fragments respectively. In addition he showed the normal 4kb Bglll fragment with probe C. A point mutation or small deletion (50bp is suspected to be present within exon 26.Patient 2 had severe haemophilia and a FVIII inhibitor of 12 units (Bethesda). DNA from patient 2 when probed with probe B revealed a novel 5.0kb TaqI fragment instead of the normal 2.2kb and 2.8kb fragments.The location of the altered Taq I restriction site within the coding region of exon 18 was confirmed with intragenomic probe pi 14.12 that includes exons 17and 18 (kindly provided by Genentech Inc.) A family study with this mutation specific fragment showed the patients sister and mother to be carriers.DNA from Patient 3 (severe haemophilia, factor VIII inhibitor 33 units) when probed independently with probes B and D revealed the absence of the normal 2.4kb and 1.4kb TaqI fragments and the generation of a novel 3.8kb TaqI fragment suggesting an alteration of the TaqI site within the coding region of exon 23.The detection of altered TaqI restriction sites in 3of our patients is further evidence that 'CG' dinucleotide sequences might be relative hot-spots for mutation when occurring within coding sequences of genes.
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Radünz, Luana Jeske, and Patrícia de Freitas Nerbas. "The Recognition and Mutation of an Ancestral Architecture." In ENSUS2023 - XI Encontro de Sustentabilidade em Projeto. Grupo de Pesquisa Virtuhab/UFSC, 2023. http://dx.doi.org/10.29183/2596-237x.ensus2023.v11.n4.p20-31.
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For different reasons and contexts, indigenous communities face adversities when trying to keep their constructive knowledge alive. This work focuses on the vernacular architecture with Brazilian cultural roots of the Guarani Mbya group, commonly overlooked by academic, political, and social circles. Its development combined bibliographical research with social participation methodologies – on-site survey through a guided tour; graphic activity with the children's audience; and discussion, with the aid of a physical model, on the traditional architecture –, addressing aspects of cultural, spatial and social organization, and inhabit and build constraints’ of the group. The result consists of a brief survey of the transmutation of this indigenous architecture over the centuries, bringing to the debate the importance of recording this constructive technical knowledge in order to keep it available for future discussions on heritage, architecture, and sustainability.
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Holmes, W. E., H. R. Lijnen, and D. Collen. "CHARACTERIZATION OFα2-ANTIPLASMIN.REACTIVE SITE VARIANTS PRODUCED BY SITE-DIRECTED MUTAGENESIS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644766.
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α2-Antiplasmin (α2AP) is the primary physiological plasmin inhibitor in human plasma. The inhibition is rapid (second order rate constants (k1) are expressed as M−1 s−1 ) (k1 = 2 × 107) and occurs as the consequence of an irreversible 1:1 stoichiometric complex formation; the exact nature of and the forces involved in complex formation are not fully understood. In fact, what makes α2AP an inhibitor, rather than simply a substrate remains unresolved. Recently, we deduced the primary structure of α2 AP from the sequence of its cDNA. 95%of this sequence was confirmed by amino acid (aa) sequence analysis of naturalα2 AP (α2 AP)? The 452 aa molecule contains 2 disulfide bonds and 4 glycosylated Asn residues, aa sequence alignment confirmed α2AP's membership in the Serpin family. The reactive site sequence as determined by NH2 - and COOH-terminal aa sequence analysis of the plasmin-modified inhibitor and the released M−r ∼ 8000 peptide is Met362-Ser363-Arg364-Met365-Ser366, P3-P2-P1-P'1-P'2, respectively.Natural and engineered P1 residue substitutions in the Serpin α2 -antitrypsin ( α2 AT) have shown altered specificities and efficiencies. To further examine the role of P and P' residues in determining Serpin specificity, in the present study we have by site-directed mutagenesis, deleted (△) the P'l-Met365 residue of a AP thereby producing a recombinant (r) inhibitor (r α2 AP△Met365) whose putative new reactive site mimics that of antithrombin III (ATIII) and a AT-Pittsburgh (Pl-Arg-P'1-Ser). A second variant was constructed (ra2AP△Arg364) in which the Pl-Arg364 residue was deleted, producing the new sequence Met362-Ser363-Met364-Ser365, containing 2 potential sites analogous to the Pl-P'l, Met-Ser reactive site of α2 AT. The variants and r α2 AP were expressed in CH0 cells, purified and compared with n α2 AP, α2AT and ATIII for the ability to inhibit plasmin, thrombin, trypsin and elastase. n α2 AP and r α2 AP had nearly identical inhibition constants and like ATIII did not inhibit neutrophil elastase. Without heparin both α2 APs and ATIII inhibited thrombin moderately (k1 = 2 to 4× 103 ). Bovine trypsin was neutralized by the α2 APs with k1 = 3 × 106 and by ATIII with k1 = 1 × 105. The α2APs inhibited plasmin (k1 = 2 ×107 ) much more efficiently than ATIII (K1 =2 × 103 ). In contrast, was a highly effective antielastase (k1 = 1 × 107 ), a poor plasmin and thrombin inhibitor ancl inhibited bovine trypsin with = 2 × 10. As reported by others, α2 AT-Pittsburg has greatly reduced antielastase activity and greatly enhanced antithrombin activity. Analysis of ra APAMet365 revealed little change in activity toward plasmin, trypsin and elastase. Thus, α2 AP has no absolute requirement for Met .in the P'l position in order to effectively inhibit plasmin and trypsin. The other P^ subsites appear to be spatially flexible as deletion of the natural P'l residue must displace them. Contrary to prediction a 20-fold decrease in antithrombin activity was observed rather than an enhanced activity. Analysis of rα2 AP△Arg364 showed that it is unreactive with plasmin, trypsin and thrombin, but that it has acquired a significant antielastase activity (k1 = 1.5 × 105). The exact PI residue(s) has not been determined but removal of the bulky basic Arg364 may have resulted in accessibility of the predicted reactive site(s) peptide bond(s) Met362-Ser363 or Met364-Ser365 to the active site cleft of elastase. α2AP'Enschede', a natural mutant with deficient antiplasmin activity, was shown to contain an Ala insertion between aa 353 and 357, 7 to 10 positions NH2-terminal to its reactive site (Holmes et al., this meeting). This mutation results in conversion of α2 AP'Enschede' from an inhibitor to a substrate that retains a high affinity for the active site of plasmin.
Reports on the topic "SITE MUTATION"
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Ohad, Itzhak, and Himadri Pakrasi. Role of Cytochrome B559 in Photoinhibition. United States Department of Agriculture, December 1995. http://dx.doi.org/10.32747/1995.7613031.bard.
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The aim of this research project was to obtain information on the role of the cytochrome b559 in the function of Photosystem-II (PSII) with special emphasis on the light induced photo inactivation of PSII and turnover of the photochemical reaction center II protein subunit RCII-D1. The major goals of this project were: 1) Isolation and sequencing of the Chlamydomonas chloroplast psbE and psbF genes encoding the cytochrome b559 a and b subunits respectively; 2) Generation of site directed mutants and testing the effect of such mutation on the function of PSII under various light conditions; 3) To obtain further information on the mechanism of the light induced degradation and replacement of the PSII core proteins. This information shall serve as a basis for the understanding of the role of the cytochrome b559 in the process of photoinhibition and recovery of photosynthetic activity as well as during low light induced turnover of the D1 protein. Unlike in other organisms in which the psbE and psbF genes encoding the a and b subunits of cytochrome b559, are part of an operon which also includes the psbL and psbJ genes, in Chlamydomonas these genes are transcribed from different regions of the chloroplast chromosome. The charge distribution of the derived amino-acid sequences of psbE and psbF gene products differs from that of the corresponding genes in other organisms as far as the rule of "positive charge in" is concerned relative to the process of the polypeptide insertion in the thylakoid membrane. However, the sum of the charges of both subunits corresponds to the above rule possibly indicating co-insertion of both subunits in the process of cytochrome b559 assembly. A plasmid designed for the introduction of site-specific mutations into the psbF gene of C. reinhardtii. was constructed. The vector consists of a DNA fragment from the chromosome of C. reinhardtii which spans the region of the psbF gene, upstream of which the spectinomycin-resistance-conferring aadA cassette was inserted. This vector was successfully used to transform wild type C. reinhardtii cells. The spectinomycin resistant strain thus obtained can grow autotrophically and does not show significant changes as compared to the wild-type strain in PSII activity. The following mutations have been introduced in the psbF gene: H23M; H23Y; W19L and W19. The replacement of H23 involved in the heme binding to M and Y was meant to permit heme binding but eventually alter some or all of the electron transport properties of the mutated cytochrome. Tryptophane W19, a strictly conserved residue, is proximal to the heme and may interact with the tetrapyrole ring. Therefore its replacement may effect the heme properties. A change to tyrosine may have a lesser affect on the potential or electron transfer rate while a replacement of W19 by leucine is meant to introduce a more prominent disturbance in these parameters. Two of the mutants, FW19L and FH23M have segregated already and are homoplasmic. The rest are still grown under selection conditions until complete segregation will be obtained. All mutants contain assembled and functional PSII exhibiting an increased sensitivity of PSII to the light. Work is still in progress for the detailed characterization of the mutants PSII properties. A tobacco mutant, S6, obtained by Maliga and coworkers harboring the F26S mutation in the b subunit was made available to us and was characterized. Measurements of PSII charge separation and recombination, polypeptide content and electron flow indicates that this mutation indeed results in light sensitivity. Presently further work is in progress in the detailed characterization of the properties of all the above mutants. Information was obtained demonstrating that photoinactivation of PSII in vivo initiates a series of progressive changes in the properties of RCII which result in an irreversible modification of the RCII-D1 protein leading to its degradation and replacement. The cleavage process of the modified RCII-D1 protein is regulated by the occupancy of the QB site of RCII by plastoquinone. Newly synthesized D1 protein is not accumulated in a stable form unless integrated in reassembled RCII. Thus the degradation of the irreversibly modified RCII-D1 protein is essential for the recovery process. The light induced degradation of the RCII-D1 protein is rapid in mutants lacking the pD1 processing protease such as in the LF-1 mutant of the unicellular alga Scenedesmus obliquus. In this case the Mn binding site of PSII is abolished, the water oxidation process is inhibited and harmful cation radicals are formed following light induced electron flow in PSII. In such mutants photo-inactivation of PSII is rapid, it is not protected by ligands binding at the QB site and the degradation of the inactivated RCII-D1 occurs rapidly also in the dark. Furthermore the degraded D1 protein can be replaced in the dark in absence of light driven redox controlled reactions. The replacement of the RCII-D1 protein involves the de novo synthesis of the precursor protein, pD1, and its processing at the C-terminus end by an unknown processing protease. In the frame of this work, a gene previously isolated and sequenced by Dr. Pakrasi's group has been identified as encoding the RCII-pD1 C-terminus processing protease in the cyanobacterium Synechocystis sp. PCC 6803. The deduced sequence of the ctpA protein shows significant similarity to the bovine, human and insect interphotoreceptor retinoid-binding proteins. Results obtained using C. reinhardtii cells exposes to low light or series of single turnover light flashes have been also obtained indicating that the process of RCII-D1 protein turnover under non-photoinactivating conditions (low light) may be related to charge recombination in RCII due to back electron flow from the semiquinone QB- to the oxidised S2,3 states of the Mn cluster involved in the water oxidation process.
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Olszewski, Neil, and David Weiss. Role of Serine/Threonine O-GlcNAc Modifications in Signaling Networks. United States Department of Agriculture, September 2010. http://dx.doi.org/10.32747/2010.7696544.bard.
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Significant evidence suggests that serine/threonine-O-linked N-acetyl glucosamine0-(GlcNAc) modifications play a central role in the regulation of plant signaling networks. Forexample, mutations in SPINDLY,) SPY (an O-GlcNAc transferase,) OGT (promote gibberellin GA) (signal transduction and inhibit cytokinin responses. In addition, mutating both Arabidopsis OGTsSEC (and SPY) causes embryo lethality. The long-term goal of this research is to elucidate the mechanism by which Arabidopsis OGTs regulate signaling networks. This project investigated the mechanisms of O-GlcNAc regulation of cytokinin and gibberellin signaling, identified additional processes regulated by this modification and investigated the regulation of SEC activity. Although SPY is a nucleocytoplasmic protein, its site of action and targets were unknown. Severalstudies suggested that SPY acted in the nucleus where it modified nuclear components such as the DELLA proteins. Using chimeric GFP-SPY fused to a nuclear-export signal or to a nuclear-import signal, we showed that cytosolic, but not nuclear SPY, regulated cytokinin and GA signaling. We also obtained evidence suggesting that GA and SPY affect cytokinin signaling via a DELLA-independent pathway. Although SEC and SPY were believed to have overlapping functions, the role of SEC in cytokinin and GA signaling was unclear. The role of SEC in cytokinin and GA responses was investigated by partially suppressing SPY expression in secplants using a synthetic Spymicro RNA miR(SPY). The possible contribution of SEC to the regulation of GA and cytokinin signaling wastest by determining the resistance of the miR spy secplants to the GA biosynthesis inhibitor paclobutrazol and to cytokinin. We found that the transgenic plants were resistant to paclobutrazol and to cytokinin, butonlyata level similar to spy. Moreover, expressing SEC under the 35S promoter in spy mutant did not complement the spy mutation. Therefore, we believe that SEC does not act with SPY to regulate GA or cytokinin responses. The cellular targets of Spy are largely unknown. We identified the transcription factor TCP15 in a two-hybrid screen for SPY-interacting proteins and showed that both TCP15 and its closely homolog TCP14 were O-GlcNAc modified by bacterially-produced SEC. The significance of the interaction between SPY and these TCPs was examined by over-expressing the minwild-type and spy-4plants. Overexpression of TCP14 or TCP15 in wild-type background produced phenotypes typical of plants with increased cytokinin and reduced GA signaling. TCP14 overexpression phenotypes were strongly suppressed in the spy background, suggesting that TCP14 and TCP15 affect cytokinin and GA signaling and that SPY activates them. In agreement with this hypothesis, we created a tcp14tcp15 double mutant and found that it has defects similar to spyplants. In animals, O-GlcNAc modification is proposed to regulate the activity of the nuclear pore. Therefore, after discovering that SEC modified a nucleoporinNUP) (that also interacts with SPY, we performed genetic experiments exploring the relationship between NUPs and SPY nupspy double mutants exhibited phenotypes consistent with SPY and NUPs functioning in common processes and nupseeds were resistant to GA biosynthesis inhibitors. All eukaryotic OGTs have a TPR domain. Deletion studies with bacterially-expressed SEC demonstrated SEC'sTPR domain inhibits SEC enzymatic activity. Since the TPR domain interacts with other proteins, we propose that regulatory proteins regulate OGT activity by binding and modulating the inhibitory activity of the TPR domain.
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Paunesku, T., M. A. Gemmell, R. Crkvenjakov, and G. E. Woloschak. Identification of a polymorphic site as a mutational site in exon VI of the mouse p53 gene. Office of Scientific and Technical Information (OSTI), July 1993. http://dx.doi.org/10.2172/10186830.
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Shai, Yechiel, Arthur Aronson, Aviah Zilberstein, and Baruch Sneh. Study of the Basis for Toxicity and Specificity of Bacillus thuringiensis d-Endotoxins. United States Department of Agriculture, January 1996. http://dx.doi.org/10.32747/1996.7573995.bard.
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The report contains three parts which summarizes the three years achievements of the three participating research groups; The Weizmann group, Tel-Aviv group and Purdue group. The firs part describes the achievements obtained by Shai's group toward the elucidation of the mechanism of membrane insertion and the structural organization of the pores formed by the Cry3A and Cry1Ac B. thuringiensis d-endotoxins. For that purpose Shai's group synthesized, fluorescently labeled and structurally and functionally characterized peptides corresponding to the seven helices that compose the pore-forming domain of Cry3A toxin, including mutants peptides and the hairpin a4G-a5 of both Cry3A and Cry 1Ac toxins composed of a4, a5 and the loop connecting a4-a5. Among the synthesized peptides were three mutated a4 helices based on site directed mutagenesis done at Aronson's group that decreased or increased Cry 1Ac toxicity. The results of these studies are consistent with a situation in which only helices a4 anda5 insert into the membrane as a helical hairpin in an antiparallel manner, while the other helices lie on the membrane surface like ribs of an umbrella (the "umbrella model"). In order to test this model Shai's group synthesized the helical hairpin a4<-->a5 of both Cry3A and Cry 1 Ac toxins, as well. Initial functional and structural studies showed direct correlation between the properties of the mutated helices and the mutated Cry1Ac. Based on Shai's findings that a4 is the second helix besides a5 that insert into the membrane, Aronson and colleagues performed extensive mutation on this helix in the CrylAc toxin, as well as in the loop connecting helices 4 and 5, and helix 3 (part two of the report). In addition, Aronson performed studies on the effect of mutations or type of insect which influence the oligomerization either the Cry 1Ab or Cry 1Ac toxins with vesicles prepared from BBMV. In the third part of the report Zilberstein's and Sneh's groups describe their studies on the three domains of Cry 1C, Cry 1E and crylAc and their interaction with the epithelial membrane of the larval midgut. In these studies they cloned all three domains and combinations of two domains, as well as cloning of the pore forming domain alone and studying its interaction with BBMV. In addition they investigated binding of Cry1E toxin and Cry1E domains to BBMV prepared from resistant (R) or sensitive larvae. Finally they initiated expression of the loop a4G<-->a5 Cry3A in E. coli to be compared with the synthetic one done by Shai's group as a basis to develop a system to express all possible pairs for structural and functional studies by Shai's group (together with Y. Shai).
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Paunesku, T., M. A. Gemmell, R. Crkvenjakov, and G. E. Woloschak. Identification of a mutational site in exon VI of the mouse p53 gene. Office of Scientific and Technical Information (OSTI), July 1993. http://dx.doi.org/10.2172/10186834.
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Wisniewski, Michael, Samir Droby, John Norelli, Dov Prusky, and Vera Hershkovitz. Genetic and transcriptomic analysis of postharvest decay resistance in Malus sieversii and the identification of pathogenicity effectors in Penicillium expansum. United States Department of Agriculture, January 2012. http://dx.doi.org/10.32747/2012.7597928.bard.
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Use of Lqh2 mutants (produced at TAU) and rNav1.2a mutants (produced at the US side) for identifying receptor site-3: Based on the fact that binding of scorpion alpha-toxins is voltage-dependent, which suggests toxin binding at the mobile voltage-sensing region, we analyzed which of the toxin bioactive domains (Core-domain or NC-domain) interacts with the DIV Gating-module of rNav1.2a. This analysis was based on the assumption that the dissociation of toxin mutants upon depolarization would vary from that of the unmodified toxin should the substitutions affect a site of interaction with the channel Gating-module. Using a series of toxin mutants (mutations at both domains) and two channel mutants that were shown to reduce the sensitivity to scorpion alpha-toxins, and by comparison of depolarization-driven dissociation of Lqh2 derivatives off their binding site at rNav1.2a mutant channels we found that the toxin Core-domain interacts with the Gating-module of DIV. Details of the experiments and results appear in Guret al (2011). Mapping receptor site 3 at Nav1.2a by extensive channel mutagenesis (Seattle): Since previous studies with photoaffinity labeling and antibody mapping implicated domains I and IV in scorpion alpha-toxin binding, Nav1.2 channel mutants containing substitutions at these extracellular regions were expressed and tested for receptor function by whole-cell voltage clamp. Of a large number of channel mutants, T1560A, F1610A, and E1613A in domain IV had ~5.9-, ~10.7-, and ~3.9-fold lower affinities for the scorpion toxin Lqh2, respectively, and mutant E1613R had 73-fold lower affinity. Toxin dissociation was accelerated by depolarization for both wild-type and mutants, and the rates of dissociation were also increased by mutations T1560A, F1610A and E1613A. In contrast, association rates for these three mutant channels at negative membrane potentials were not significantly changed and were not voltage-dependent. These results indicated that Thr1560 in the S1-S2 loop, Phe1610 in the S3 segment, and Glu1613 in the S3-S4 loop in domain IV participate in toxin binding. T393A in the SS2-S6 loop in domain I also showed a ~3.4-fold lower affinity for Lqh2, indicating that this extracellular loop may form a secondary component of the toxin binding site. Analysis with the Rosetta-Membrane algorithm revealed a three-dimensional model of Lqh2 binding to the voltage sensor in a resting state. In this model, amino acid residues in an extracellular cleft formed by the S1-S2 and S3-S4 loops in domain IV that are important for toxin binding interact with amino acid residues on two faces of the wedge-shaped Lqh2 molecule that are important for toxin action. The conserved gating charges in the S4 transmembrane segment are in an inward position and likely form ion pairs with negatively charged amino acid residues in the S2 and S3 segments (Wang et al 2011; Gurevitz 2012; Gurevitzet al 2013).
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Varma, M. N., V. P. Bond, and G. Matthews. Hit-size effectiveness theory applied to high doses of low LET radiation for pink mutations in Tradescantia. Office of Scientific and Technical Information (OSTI), January 1985. http://dx.doi.org/10.2172/5612375.
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Zheng, Jiaxi, and Haihua Yang. Clinical Benefits of Immune Checkpoint Inhibitors and Predictive Value of Tumor Mutation Burden in Hepatocellular Carcinoma: A Systematic Review and Meta-Analysis. INPLASY - International Platform of Registered Systematic Review and Meta-analysis Protocols, January 2022. http://dx.doi.org/10.37766/inplasy2022.1.0008.
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Review question / Objective: Is immunotherapy associated with beneficial clinical outcomes for hepatocellular carcinoma (HCC) and how can combination immunotherapy be deployed to produce the best benefit? Is tumor mutation burden (TMB) a predictive biomarker for immune‐checkpoint inhibitors? Condition being studied: To this date, about 50 single-arm clinical trials and several randomized control trials (RCTs) presented final or interim results of investigations on the efficacy of PD-1/PD-L1 inhibitors for advanced HCC. In the CheckMate 459, IMbrave 050, and ORIENT-32, immunotherapies were found to significantly improve progression-free survival (PFS) and overall survival (OS) compared with sorafenib (a tyrosine-kinase inhibitor, as standard systemic treatment) in patients with advanced hepatocellular carcinoma. However, these clinical trials were different on clinical phases, sample size, and response evaluation criteria, and inconsistent clinical outcomes were shown in several trials.
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Paran, Ilan, and Allen Van Deynze. Regulation of pepper fruit color, chloroplasts development and their importance in fruit quality. United States Department of Agriculture, January 2014. http://dx.doi.org/10.32747/2014.7598173.bard.
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Pepper exhibits large natural variation in chlorophyll content in the immature fruit. To dissect the genetic and molecular basis of this variation, we conducted QTL mapping for chlorophyll content in a cross between light and dark green-fruited parents, PI 152225 and 1154. Two major QTLs, pc1 and pc10, that control chlorophyll content by modulation of chloroplast compartment size in a fruit-specific manner were detected in chromosomes 1 and 10, respectively. The pepper homolog of GOLDEN2- LIKE transcription factor (CaGLK2) was found as underlying pc10, similar to its effect on tomato fruit chloroplast development. A candidate gene for pc1was found as controlling chlorophyll content in pepper by the modulation of chloroplast size and number. Fine mapping of pc1 aided by bulked DNA and RNA-seq analyses enabled the identification of a zinc finger transcription factor LOL1 (LSD-One-Like 1) as a candidate gene underlying pc1. LOL1 is a positive regulator of oxidative stress- induced cell death in Arabidopsis. However, over expression of the rice ortholog resulted in an increase of chlorophyll content. Interestingly, CaAPRR2 that is linked to the QTL and was found to affect immature pepper fruit color in a previous study, did not have a significant effect on chlorophyll content in the present study. Verification of the candidate's function was done by generating CRISPR/Cas9 knockout mutants of the orthologues tomato gene, while its knockout experiment in pepper by genome editing is under progress. Phenotypic similarity as a consequence of disrupting the transcription factor in both pepper and tomato indicated its functional conservation in controlling chlorophyll content in the Solanaceae. A limited sequence diversity study indicated that null mutations in CaLOL1 and its putative interactorCaMIP1 are present in C. chinensebut not in C. annuum. Combinations of mutations in CaLOL1, CaMIP1, CaGLK2 and CaAPRR2 are required for the creation of the extreme variation in chlorophyll content in Capsicum.
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Locy, Robert D., Hillel Fromm, Joe H. Cherry, and Narendra K. Singh. Regulation of Arabidopsis Glutamate Decarboxylase in Response to Heat Stress: Modulation of Enzyme Activity and Gene Expression. United States Department of Agriculture, January 2001. http://dx.doi.org/10.32747/2001.7575288.bard.
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Most plants accumulate the nonprotein amino acid, g-aminobutyric acid (GABA), in response to heat stress. GABA is made from glutamate in a reaction catalyzed by glutamate decarboxylase (GAD), an enzyme that has been shown by the Israeli PI to be a calmodulin (CaM) binding protein whose activity is regulated in vitro by calcium and CaM. In Arabidopsis there are at least 5 GAD genes, two isoforms of GAD, GAD1 and GAD2, are known to be expressed, both of which appear to be calmodulin-binding proteins. The role of GABA accumulation in stress tolerance remains unclear, and thus the objectives of the proposed work are intended to clarify the possible roles of GABA in stress tolerance by studying the factors which regulate the activity of GAD in vivo. Our intent was to demonstrate the factors that mediate the expression of GAD activity by analyzing the promoters of the GAD1 and GAD2 genes, to determine the role of stress induced calcium signaling in the regulation of GAD activity, to investigate the role of phosphorylation of the CaM-binding domain in the regulation of GAD activity, and to investigate whether ABA signaling could be involved in GAD regulation via the following set of original Project Objectives: 1. Construction of chimeric GAD1 and GAD2 promoter/reporter gene fusions and their utilization for determining cell-specific expression of GAD genes in Arabidopsis. 2. Utilizing transgenic plants harboring chimeric GAD1 promoter-luciferase constructs for isolating mutants in genes controlling GAD1 gene activation in response to heat shock. 3. Assess the role of Ca2+/CaM in the regulation of GAD activity in vivo in Arabidopsis. 4. Study the possible phosphorylation of GAD as a means of regulation of GAD activity. 5. Utilize ABA mutants of Arabidopsis to assess the involvement of this phytohormone in GAD activation by stress stimuli. The major conclusions of Objective 1 was that GAD1 was strongly expressed in the elongating region of the root, while GAD2 was mainly expressed along the phloem in both roots and shoots. In addition, GAD activity was found not to be transcriptionally regulated in response to heat stress. Subsequently, The Israeli side obtained a GAD1 knockout mutation, and in light of the objective 1 results it was determined that characterization of this knockout mutation would contribute more to the project than the proposed Objective 2. The major conclusion of Objective 3 is that heat-stress-induced changes in GAD activity can be explained by heat-stress-induced changes in cytosolic calcium levels. No evidence that GAD activity was transcriptionally or translationally regulated or that protein phosphorylation was involved in GAD regulation (objective 4) was obtained. Previously published data by others showing that in wheat roots ABA regulated GABA accumulation proved not to be the case in Arabidopsis (Objective 5). Consequently, we put the remaining effort in the project into the selection of mutants related to temperature adaptation and GABA utilization and attempting to characterize events resulting from GABA accumulation. A set of 3 heat sensitive mutants that appear to have GABA related mutations have been isolated and partially characterized, and a study linking GABA accumulation to growth stimulation and altered nitrate assimilation were conducted. By providing a better understanding of how GAD activity was and was not regulated in vivo, we have ruled out the use of certain genes for genetically engineering thermotolerance, and suggested other areas of endeavor related to the thrust of the project that may be more likely approaches to genetically engineering thermotolerance.