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1
Wilson, Lindsay Anne. "The enterobacterial repeated intergenic consensus (ERIC) sequence." Thesis, University of Nottingham, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.342441.
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Mokin, Sergey. "Measuring deviation from a deeply conserved consensus in protein multiple sequence alignments." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=21956.
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Proteins across species show variable degrees of conservation. Different patterns of conservation in the columns of an alignment indicate different evolutionary pressures on sequences. Protein conservation analysis is useful for a wide variety of applications, including disease mutation assessment, pseudogene analysis and functional residue prediction. This study describes a novel measure of column conservation in protein multiple sequence alignments (‘MSA'), and the application of this measure to calculate statistical deviation from alignment consensus (‘SDAC'). We have assessed SDAC for two case studies of sequences: (a) putative pseudogenes in Mycobacteria, and (b) young lineage-specific retrotransposed sequences in the human and mouse genomes. In the procedure, we rank residue positions for deep conservation, and evaluate statistically significant violations from MSA consensus. Novel conservation measure clearly indicated a variable degree of physiochemical conservation for a given column entropy. That, in turn, enabled us to detect deviations from physiochemical consensus in a protein MSA, which are not found by entropy measures.<br>D'une espèce à l'autre, des variations peuvent survenir dans la composition des protéines. Les tendances suivies par les colonnes d'un alignement de séquences multiples reflètent les différentes pressions évolutionnaires imposes sur les séquences. Les analyses de conservation de protéines sont utiles à plusieurs fins, comme dans l'évaluation des mutations de maladies, l'analyse de pseudogenes ainsi que les prédictions fonctionnelles de résidus. Cette étude décrit une nouvelle mesure de conservation de colonnes pour les analyses d'alignement de séquences multiples. De plus, nous décrivons l'utilisation de cette nouvelle mesure pour calculer la déviation statistique avec un consensus d'alignement. Nous avons utilisé cette mesure pour deux études cas de séquence : (a) Celle de pseudogenes putatifs du Mycobactérie, et (b) Celle de jeunes séquences spécifiques a certains lignages rétrotransposés dans les génomes humains et souris. Ce faisant, nous avons classifié les positions de résidus hautement conservés et avons évalué les cas ou d'importantes variations existent avec les consensus des alignements de séquences multiples. Cette nouvelle échelle de conservation indique qu'il existe un degré variable de conservation physiochimique pour une entropie fixe des colonnes. En retour, ceci nous permet de détecter les variations physiochimiques des consensus d'une colonne qui ne serait autrement pas détecté par des mesures d'entropie.
3
Kamura, Eri. "Exploring the Methylation Status of RAI1 and the RAI1 Consensus Binding Sequence." VCU Scholars Compass, 2009. http://scholarscompass.vcu.edu/etd/1891.
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Smith Magenis Syndrome (SMS) is a multiple congenital anomalies/ mental retardation disorder caused by deletion or mutation of the RAI1 gene on chromosome 17p11.2. The majority of patients with SMS phenotypes have a deletion or mutation of RAI1. However, some patients have been observed with SMS-like phenotypes and yet have no deletions or mutations in the RAI1 gene. One possible explanation could be aberrant methylation of RAI1 since RAI1 is present and yet may be silenced. In order to study this possibility, patient cell lines were treated with 5-Aza-2’-deoxycytidine. RNA was extracted and real-time PCR was used to check the RAI1 expression status on the cells. RAI1 is thought to be a transcription factor, but the DNA binding sequence is still unknown. Sequences from ChIP-chip data were compared to identify a consensus sequence. One gene which contained this consensus sequence was the chemokine-like receptor-1 gene (CMKLR1), which was investigated by luciferase assay. CMKLR1 showed upregulation when co-transfected with RAI1.
4
Babst, Scheán. "Mitochondrial DNA consensus sequence for the Tswana population of South Africa / Scheán Babst." Thesis, North-West University, 2012. http://hdl.handle.net/10394/9110.
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Evolutionary studies are critical in eliciting the fundamental phylogeny within and among
populations of living organisms. Genetic diversity is displayed in human mitochondrial DNA
(mtDNA) as haplogroups that consist of shared mutations, which are carried to the
following generation through the maternal lineage. The current haplogroup hierarchies
commonly used to describe and compare the genetic diversity of global human
populations are based on the available mtDNA sequence variation datasets of numerous
continent-specific populations. The description of mtDNA variation in human populations is
furthermore of importance, as it allows the identification of population-specific genetic
variation that has an effect on gene function, as well as on adaptation and susceptibility to
disease. Owing to the limited amount of available mtDNA variation data from the
numerous African populations currently residing in Africa, a lack of genetic diversity data
exists for the determination of a sufficient baseline standard sequence representing the
genetic variation present in African populations and thus also for a representative African
haplogroup hierarchy.
In this study, the mtDNA variation of 50 Tswana-speaking individuals from South Africa
was determined and a novel Tswana consensus sequence was constructed to contribute
to the urgent need for information of the mtDNA variation present in African populations.
The consensus mtDNA sequence variation data obtained through this analysis should be
regarded as a baseline for the observed sequence variance and genetic diversity of the
maternal ancestral genetic pool of a Bantu-speaking population of South Africa.
This study therefore contributes novel information regarding the mitochondrial genetic
diversity of a South African Tswana-speaking population to the current body of literature.
The results of this study provide strong evidence to support the ancient nature of African
haplogroups and also provide evidence in support of the presence of Khoi-San maternal
ancestry in the origins of the current Bantu-speaking populations of southern Africa. In
addition, the observed sequence variation contributes to the current haplogroup hierarchy
of African lineages and provides information in support of the previously reported distinct
phylogenetic relationship between individuals of African and non-African origin, thereby
explaining the high level of genetic diversity among and between African populations.<br>Thesis (PhD (Biochemistry))--North-West University, Potchefstroom Campus, 2013
5
Mouton, Christa. "Mitochondrial genome consensus sequence for the South African Khoi-San population / Christa Mouton." Thesis, North-West University, 2003. http://hdl.handle.net/10394/9618.
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Maternal inheritance and the absence of recombination have contributed to mitochondrial
deoxyribonucleic acid (mtDNA) being utilised to study human evolution. This, together with
an increased mutation rate in mtDNA, provides information about the most recent common
ancestor of modern humans.
Previous studies suggested that Africa harbours the highest mtDNA diversity, supporting
an out-of-Africa hypothesis for modern human evolution. From subsequent studies it was
suggested that the Khoi-San population, in particularly the !Kung, cluster at the deepest
root of the global phylogenetic tree.
The Cambridge reference sequence is used worldwide in mitochondrial studies as a
reference. However, recent studies have observed discrepancies from this sequence,
which were confirmed by reanalysis.
During this investigation the complete mitochondrial sequences of 13 !Kung individuals
were determined. From phylogenetic analyses their clustering in the African LO-Iineage
was revealed. The evolutionary rate of the derived sequences was investigated through
statistical analysis and the hypothesis of neutral evolution was rejected. Pairwise
nucleotide distribution suggested that sequences representing haplogroups LO, L 1 and L2
are examples of populations that were of stable population size for a long time. However,
L3 was suggested to have been subjected to population expansion, in support of the
out-of-Africa theory of evolution.
From the comparative analysis of the 13 !Kung sequences with an LO-specific haplogroup
tree it was observed that the 13 individuals clustered in two main groups. Ten individuals
were added to one branch of the phylogenetic tree, revealing further branching, while three
individuals were added to the terminal branches of another tree topology. A consensus
sequence was derived from the 13 Khoi-San sequences, which was 99.25% similar to
each of the sequences. This sequence could be utilised to investigate evolution of the
mitochondrial genome over time as well as to evaluate the pathogenicity of mutations in
patients.<br>MSc (Biochemistry) North-West University, Potchefstroom Campus, 2004
6
Wynn, Anna. "Four differentially expressed cDNAs containing the Rebers-Riddiford consensus sequence in Callinectes sapidus /." Electronic version (PDF), 2004. http://dl.uncw.edu/etd/2004/wynna/annawynn.pdf.
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Ng, Harald. "Distributed Consensus: Performance Comparison of Paxos and Raft." Thesis, KTH, Skolan för elektroteknik och datavetenskap (EECS), 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-281973.
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With the growth of the internet, distributed systems have become increasingly important in order to provide more available and scalable applications. Con- sensus is a fundamental problem in distributed systems where multiple pro- cesses have to agree on the same proposed value in the presence of partial failures. Distributed consensus allows for building various applications such as lock services, configuration manager services or distributed databases.Two well-known consensus algorithms for building distributed logs are Multi-Paxos and Raft. Multi-Paxos was published almost three decades before Raft and gained a lot of popularity. However, critics of Multi-Paxos consider it difficult to understand. Raft was therefore published with the motivation of being an easily understood consensus algorithm. The Raft algorithm shares similar characteristics with a practical version of Multi-Paxos called Leader- based Sequence Paxos. However, the algorithms differ in important aspects such as leader election and reconfiguration.Existing work mainly compares Multi-Paxos and Raft in theory, but there is a lack of performance comparisons in practice. Hence, prototypes of Leader- based Sequence Paxos and Raft have been designed and implemented in this thesis. The prototypes were implemented using the Rust programming lan- guage and the message-passing framework Kompact and then benchmarked in real-world scenarios to compare the performance of Leader-based Sequence Paxos and Raft.The results show that Leader-based Sequence Paxos and Raft have simi- lar performance in geographically distributed deployments. However, the un- predictable leader election in Raft could greatly affect the performance if the elected leader is in an undesired location. In our experiments, the location of the Raft leader affected the average throughput by up to 35%. Furthermore, the results indicate that implementation details could have a significant impact on performance even in the parts where the algorithms are similar. By batch- ing messages more efficiently, Leader-based Sequence Paxos achieved up to 17% higher average throughput than Raft.<br>Med tillväxten av internet har distribuerade system blivit allt mer viktiga för att bygga mer tillgängliga och skalbara applikationer. Konsensus är ett funda- mentalt problem i distribuerade system där flera processer ska komma överens om samma föreslagna värde, samtidigt som partiella fel kan ske. Distribuerad konsensus kan appliceras till olika användningsomården som låstjänster, kon- figurationshanterare och distribuerade databaser.Två välkända konsensusalgoritmer för att bygga distribuerade loggar är Multi-Paxos och Raft. Multi-Paxos publicerades nästintill tre årtionden före Raft och blev populär. Men kritiker av Multi-Paxos anser att algoritmen är svår att förstå. Av denna anledning publicerades Raft med motivationen att vara en konsensusalgoritm som är enkel att förstå. Raft delar likheter med Leader- based Sequence Paxos, en praktisk version av Multi-Paxos. Dock skiljer sig algoritmerna i viktiga aspekter som leaderval och rekonfigurering.Befintliga arbeten jämför i huvudsak Multi-Paxos och Raft i teorin, men det saknas jämförelse av prestandan i praktiken. Av denna anledning har pro- totyper av Leader-based Sequence Paxos och Raft blivit designade och imple- menterade i denna avhandling. Dessa prototyper implementerades i program- meringsspråket Rust och message-passing ramverket Kompact, som sedan tes- tades i verkliga situationer för att jämföra Leader-based Sequence Paxos och Raft.Resultaten visar att Leader-based Sequence Paxos och Raft har liknande prestanda i geografiskt distribuerade sammanhang. Dock kan det oförutsäga- bara ledarvalet i Raft påverka prestandan avsevärt ifall den valde ledaren befin- ner sig på en oönskad plats. I våra experiment påverkade Raft ledarens plats den genomsnittliga kapaciteten med upp till 35%. Resultaten visar även att implementationsdetaljer kan ha en signifikant effekt på prestandan även i de delar där algoritmerna är liknande. Genom att sammanfoga meddelanden mer effektivt uppnådde Leader-based Sequence Paxos 17% högre genomsnittlig kapacitet än Raft.
8
Wen, Meimei. "Structural studies of a consensus sequence peptide (CSP) ABAB of apolipoproteins through NMR spectroscopy." Thesis, Boston University, 2013. https://hdl.handle.net/2144/11084.
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Thesis (Ph.D.)--Boston University<br>The apolipoproteins play critical roles in lipid transport, lipid metabolism and the pathophysiology of dyslipoproteinemias, most importantly atherosclerosis. ApoA-1 is a representative member of the family of exchangeable apolipoproteins and the major apolipoprotein of high density lipoprotein (HDL). HDL is responsible for the pathway of reverse cholesterol transport and the only particle capable of removing cholesterol from peripheral cells for transport to the liver.
The sequences ofthe exchangeable apolipoproteins contain 11/22 residue tandem sequence repeats forming amphipathic α-helices that are believed to be responsible for lipid binding. The consensus sequence peptide (CSP) for this repeat was derived based on the characteristic residue distribution of the exchangeable apolipoproteins. The derived consensus sequence containing motifs A, (PLAEELRARLR), and B, (AQLEELRERLG), represent an idealized lipid binding model and fundamental structural motif of the exchangeable apolipoproteins.
The recombinant CSP-ABAB peptide was successfully expressed in E. coli and purified. Circular dichroism showed that CSP-ABAB is ~62% α-helical, i.e.~27 residues of 44 residues are in helical conformation. The CSP-ABAB peptide was successfully 15N, 13C labeled and the detailed tertiary structure was explored by NMR spectroscopy. The peptide's backbone and side-chain resonances were successfully assigned and ten water refined structural conformers of CSP-ABAB were generated.
The ten structural conformers all employ anti-parallel helical conformation in solution. Hydrophobic inter-helical interactions play a major role to stabilize the antiparallel helical hairpin conformation. There are also intra-/inter-helical salt bridges present on the surface of the CSP-ABAB molecule providing additional stabilization. The structural features of the NMR structures suggest a lipid binding model of CSP-ABAB.
When lipids are introduced, the exposed hydrophobic ridge contributed by the twelve leucine residues firstly bind to the lipids. At the same time, a hydrophobic concave surface created by the four alanine residues at the center of the interface is accessed by the introduced lipids. These two steps open the anti-parallel helical hairpin conformation to form a fully extended α-helix. Similar hydrophobic inter-helical stabilization interactions and new intra-/inter-helical salt bridges between two different CSP-ABAB molecules are reformed to stabilize the 'double-belt' arrangement.
This lipid binding model of CSP-ABAB sheds light on the lipid binding of apoA-I and the mechanism of HDL formation.
9
McMillen, Lyle, and l. mcmillen@sct gu edu au. "Isolation and Characterisation of the 5'-Nucleotidase from Escherichia coli." Griffith University. School of Biomolecular and Biomedical Science, 2001. http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20030226.153545.
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Escherichia coli 5'-nucleotidase is a periplasmically localised enzyme capable of hydrolysing a broad range of substrates, including all 5'-ribo- and 5'-deoxyribonucleotides, uridine diphosphate sugars, and a number of synthetic substrates such as bis (r-nitrophenyl) phosphate. The enzyme has been shown to contain at least one zinc ion following purification, and to have two metal binding sites in the catalytic cleft. 5'-Nucleotidase activity is significantly stimulated by the addition of particular divalent metal ions, most notably cobalt which results in a 30-50 fold increase in activity. Significant sequence homology between the E. coli 5'-nucleotidase and members of the Ser/Thr protein phosphatase family in the catalytic site has lead to 5'-nucleotidase being included in this protein family. This thesis describes the development of a rapid purification methodology for milligram quantities of 5'-nucleotidase, and the investigation of a number of physical and biochemical properties of the enzyme with the aim of comparing these properties to those of certain catalytic site mutants. The molecular weight of the mature protein was estimated as 58219 daltons, with a specific activity for 5'-AMP, in the presence of 4 mM Co2+ and 13 mM Ca2+ at pH 6.0, of 730 mmol/min/mg. The presence of up to two zinc ions associated with the purified enzyme was observed using ICP-ES analysis, suggesting both metal ion binding sites are occupied by zinc in vivo, and some degree of displacement of zinc by cobalt could be observed. Mass spectrometry data, gathered at 60 and 70 mS orifice potential, suggested the presence of a small proportion of material with a mass 118 to 130 daltons greater than the main 5'-nucleotidase mass estimation. This study suggests that this mass difference, only evident at the lower orificepotential, is due to the presence of two zinc ions closely associated with 5'-nucleotidase. To account for the observed high level of activation of 5'-nucleotidase activity by particular divalent metal ions, this thesis describes a proposed model in which these divalent ions may displace the zinc ion at one of the metal ion binding sites. This displacement only occurs at one of the two metal ion binding sites, with the other metal binding site retaining the zinc ion already present. Studies with purified enzyme, each with a single amino acid substitution, lend support to this hypothesis and suggest the identity of the metal ion binding site at which displacement occurs. Seven key catalytic site residues (Asp-41, His-43, Asp-84, His-117, Glu-118, His-217 and His-252) were selected on the basis of sequence conservation within the Ser/Thr protein phosphatases and 5'-nucleotidases. X-ray crystallographic data published by others during this study implicated five of the selected residues (Asp-41, His-43, Asp-84, His-217 and His-252) directly in metal ion binding, including two residues from each metal ion binding site and one directly involved in both sites (Asp-84). The remaining two residues (His-117 and Glu-118) are highly conserved but were not thought to play direct roles in metal ion binding. The seven selected residues were modified by site-directed mutagenesis, and the effect of the amino acid substitutions upon the kinetic properties of 5'-nucleotidase activity was determined. Residues hypothesised to be involved in metal ion displacement, and subsequent activation of 5'-nucleotidase activity, were identified by reductions in metal ion affinity and increased levels of activation by cobalt compared to the wild type 5'-nucleotidase. This study suggests that the metal binding site, M2, that includes residues Asp-84, His-217 and His-252, is involved in metal ion displacement, while the other metal binding site, M1, is not. This, in turn, suggests the metal binding sites are functionally non-equivalent and kinetically distinct. No residues were identified in this study as playing significant roles in substrate binding, as there was no significant reduction observed in affinity for 5'-AMP observed in any of the catalytic site mutants.
10
McMillen, Lyle. "Isolation and Characterisation of the 5'-Nucleotidase from Escherichia coli." Thesis, Griffith University, 2001. http://hdl.handle.net/10072/366487.
Texto completoResumen
Escherichia coli 5'-nucleotidase is a periplasmically localised enzyme capable of hydrolysing a broad range of substrates, including all 5'-ribo- and 5'-deoxyribonucleotides, uridine diphosphate sugars, and a number of synthetic substrates such as bis (r-nitrophenyl) phosphate. The enzyme has been shown to contain at least one zinc ion following purification, and to have two metal binding sites in the catalytic cleft. 5'-Nucleotidase activity is significantly stimulated by the addition of particular divalent metal ions, most notably cobalt which results in a 30-50 fold increase in activity. Significant sequence homology between the E. coli 5'-nucleotidase and members of the Ser/Thr protein phosphatase family in the catalytic site has lead to 5'-nucleotidase being included in this protein family. This thesis describes the development of a rapid purification methodology for milligram quantities of 5'-nucleotidase, and the investigation of a number of physical and biochemical properties of the enzyme with the aim of comparing these properties to those of certain catalytic site mutants. The molecular weight of the mature protein was estimated as 58219 daltons, with a specific activity for 5'-AMP, in the presence of 4 mM Co2+ and 13 mM Ca2+ at pH 6.0, of 730 mmol/min/mg. The presence of up to two zinc ions associated with the purified enzyme was observed using ICP-ES analysis, suggesting both metal ion binding sites are occupied by zinc in vivo, and some degree of displacement of zinc by cobalt could be observed. Mass spectrometry data, gathered at 60 and 70 mS orifice potential, suggested the presence of a small proportion of material with a mass 118 to 130 daltons greater than the main 5'-nucleotidase mass estimation. This study suggests that this mass difference, only evident at the lower orificepotential, is due to the presence of two zinc ions closely associated with 5'-nucleotidase. To account for the observed high level of activation of 5'-nucleotidase activity by particular divalent metal ions, this thesis describes a proposed model in which these divalent ions may displace the zinc ion at one of the metal ion binding sites. This displacement only occurs at one of the two metal ion binding sites, with the other metal binding site retaining the zinc ion already present. Studies with purified enzyme, each with a single amino acid substitution, lend support to this hypothesis and suggest the identity of the metal ion binding site at which displacement occurs. Seven key catalytic site residues (Asp-41, His-43, Asp-84, His-117, Glu-118, His-217 and His-252) were selected on the basis of sequence conservation within the Ser/Thr protein phosphatases and 5'-nucleotidases. X-ray crystallographic data published by others during this study implicated five of the selected residues (Asp-41, His-43, Asp-84, His-217 and His-252) directly in metal ion binding, including two residues from each metal ion binding site and one directly involved in both sites (Asp-84). The remaining two residues (His-117 and Glu-118) are highly conserved but were not thought to play direct roles in metal ion binding. The seven selected residues were modified by site-directed mutagenesis, and the effect of the amino acid substitutions upon the kinetic properties of 5'-nucleotidase activity was determined. Residues hypothesised to be involved in metal ion displacement, and subsequent activation of 5'-nucleotidase activity, were identified by reductions in metal ion affinity and increased levels of activation by cobalt compared to the wild type 5'-nucleotidase. This study suggests that the metal binding site, M2, that includes residues Asp-84, His-217 and His-252, is involved in metal ion displacement, while the other metal binding site, M1, is not. This, in turn, suggests the metal binding sites are functionally non-equivalent and kinetically distinct. No residues were identified in this study as playing significant roles in substrate binding, as there was no significant reduction observed in affinity for 5'-AMP observed in any of the catalytic site mutants.<br>Thesis (PhD Doctorate)<br>Doctor of Philosophy (PhD)<br>School of Biomolecular and Biomedical Sciences<br>Science, Environment, Engineering and Technology<br>Full Text
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Brown, Andrew S. "Identification of a phospho-hnRNP E1 Nucleic Acid Consensus Sequence Mediating Epithelial to Mesenchymal Transition." Kent State University / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=kent1437943957.
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Mohan, Sidharth. "Consensus, Correlation And Combinatorics Based Approaches In Engineering And Exploring Triosephosphate Isomerase Stability." The Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1503054678218166.
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Li, Chunjie. "Development of a FRET biosensor for ROCK based on a consensus substrate sequence identified by KISS technology." 京都大学 (Kyoto University), 2017. http://hdl.handle.net/2433/225755.
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Koekemoer, Michelle. "Construction of a mitochondrial consensus sequence for the Khoi-San population of Southern Africa / by Michelle Koekemoer." Thesis, North-West University, 2010. http://hdl.handle.net/10394/4397.
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The revised Cambridge Reference Sequence (rCRS) is used as a standard for the human mitochondrial DNA (mtDNA) sequence in studies of human evolution and the identification of disease-causing mutations. Due to the large number of differences observed between the rCRS and mitochondrial sequences obtained from individuals of African descent, it is frequently difficult to differentiate between alterations that are population-specific or have possible pathological significance. To address this problem, two human consensus sequences consisting of mitochondrial sequences from different continents and different haplogroups have been constructed. However, combining data from different continents and haplogroups led to a loss of variation in the human mitochondrial consensus sequences. This can be countered by using an African reference sequence, of which two sequences are currently available, namely NC_001807 (L3a1) and D38112 (L0c2). However, these sequences are not representative of the most ancient African populations (i.e. hunter-gatherers) or haplogroups (i.e. L0).
In the current investigation, the complete mitochondrial genome sequences of 30 Khoi-San individuals from southern Africa were determined. Twenty-two of these Khoi-San sequences, which belong to the L0a and L0b sub-haplogroups, were combined with 13 L0 Khoi-San sequences generated previously to compile a consensus sequence for the Southern African Khoi-San population. This Khoi-San consensus sequence will represent the first example of an African population-specific consensus sequence.
The results presented in the current investigation provide support for previous findings regarding the existence of a high level of genetic variation in the mtDNA of the Khoi-San population, as well as the ancient character of the Khoi-San population. In addition, it offers novel insights into the complexity of the L0 haplogroup within the Khoi-San population and the African population as a whole. The high level of genetic variation and increased age of mtDNA lineages in the Southern African Khoi-San population compared to other populations, support the use of the Khoi-San consensus sequence, alone or in conjunction with the rCRS, as a standard in studies of human evolution and mitochondrial disease.<br>Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2010.
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Barozzi, I. G. "OVERLAPPING SEQUENCE FEATURES OF MAMMALIAN ENHANCERS COORDINATELY CONTROL ENGAGEMENT OF TRANSCRIPTION FACTOR CONSENSUS SITES AND NUCLEOSOMAL OCCUPANCY." Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/234131.
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In mammalian cells transcription factors (TFs) bind only to a small fraction of the available consensus sites in the genome. In particular, they prefer sites embedded in regions of computationally predicted high nucleosomal occupancy. This is compatible with non-exclusive mechanisms of nucleosome-driven TF-binding and nucleosome-mediated masking of TF binding sites, suggesting that TFs, and in particular pioneers, must overcome a strong barrier in order to engage binding. Exploiting the available information for the hematopoietic master regulator Pu.1, we applied machine learning approaches and uncovered the sequence-encoded information that discriminates engaged from non-engaged genomic consensus sites. We identified a minimal set of features which predicts Pu.1 binding with 78% accuracy, among which sequence determinants able to drive nucleosome occupancy were found. Consistent with this, while Pu.1 maintained nucleosome depletion at many thousand cell type-specific enhancers in macrophages, these site are otherwise occupied by nucleosomes in other cell types and in in vitro reconstituted chromatin. As predicted, engaged consensus sites showed higher sequence-encoded nucleosome occupancy compared to the myriad of non-occupied (and likely non-functional) consensus sites that randomly occur in mammalian genomes. The same sequence features selected in machine learning also explains up to 45% of the variability observed in the nucleosome occupancy in cells where Pu.1 is not expressed (a performance equal or better than what achieved by ad hoc models), suggesting that the same information contributes to nucleosome occupancy and positioning. These data reveal a basic organizational principle of mammalian enhancers whereby TF-engagement at its consensus sites and nucleosome occupancy are coordinately controlled by overlapping sequence features. This model also suggests that co-evolution of these features may be crucial to ensure cell-type specific enhancer activation. The nucleosomal patterns at Pu.1-bound sites in macrophages were further characterized, uncovering distinct subtypes with different DNA sequence composition, which mirror distinctive nucleosomal configurations either in the presence or in the absence of Pu.1.
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Rossl, Anthony. "A Synthetic Acetylation Substrate to Study Gcn5 Targeting and Function in Yeast." Thesis, Université d'Ottawa / University of Ottawa, 2018. http://hdl.handle.net/10393/38300.
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Acetylation was previously thought to occur exclusively on histones, but recent high-throughput screens have identified thousands of non-histone substrates. Despite the identification of these sites, little is known about how these acetyltransferase enzymes target their substrates. Gcn5 is the catalytic acetyltransferase found within the highly conserved SAGA complex. Recently, a member of this complex, Ada2, was found to impact Gcn5 substrate selection. In the yeast model organism Saccharomyces cerevisiae, a synthetic substrate developed from a proposed Gcn5-specific consensus sequence is used to identify regulators of Gcn5 substrate selection.
This work is the first to demonstrate that addition of a consensus sequence is enough to confer acetylation of a non-substrate. With this method, Ada3 was identified as a key regulator, and acetylome profiling identified novel targets for Gcn5 dependent acetylation specifically regulated by Ada3. This system could be adapted for other acetyltransferases to identify regulators of substrate selection.
17
Tanaka, Emi. "Statistical Methods for Improving Motif Evaluation." Thesis, The University of Sydney, 2014. http://hdl.handle.net/2123/13922.
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Gene regulation, especially cis-regulation of gene expression by the binding of transcription factors, is a critical component of cellular physiology. Transcription regulation is heavily influenced by the binding of transcription factors, and as such, it is of great interest to characterise these binding sites. The binding sites of a transcription factor are collectively referred to as a regulatory motif. Recent advancement in sequencing technology generated vast amounts of biological data. Thus computational tools are required to process and analyse this massive information. In particular, computational tools were developed to search for over-represented words among a set of co-regulated sequences. Such tools would be somewhat incomplete without a statistical analysis that allows researchers to discern between real biological significant sites and random artefacts. By analogy, it is difficult to imagine evaluating a BLAST result without its accompanying E-value. Of the many motif finders, MEME, with over 9000 unique users recorded in the first half of 2013, is one of the most popular motif finding tools available. Currently MEME evaluates its candidate motifs using an extension of BLAST's E-value to the motif finding context. Ng et al. (2006) previously showed the drawbacks of MEME's current significance evaluation scheme, however because MEME relies on the same E-value to internally rank competing candidate motifs, the alternative evaluation offered by Keich and Ng (2007) was not a practical substitute. Here we offer a two-tiered significance analysis that can replace the E-value in selecting the best candidate motif as well as in evaluating its overall statistical significance. We show that our new approach substantially improves MEME's motif finding performance and also provides the user with a reliable significance analysis. In addition, for large input sets our new approach is faster than the currently implemented E-value analysis. After applying a motif finder to a set of co-regulated DNA sequences, researchers often are interested to know whether the reported putative motif is similar to any known motif. While several tools have been designed for this task, Habib et al. (2008) pointed out that the scores that are commonly used for measuring similarity between motifs do not distinguish between a good alignment of two informative columns (say, all-A) and one of two uninformative columns. This observation explains why motif comparison tools such as Tomtom occasionally return an alignment of uninformative columns which is clearly spurious. To address this distinguishability problem Habib et al. (2008) suggested a new score, the BLiC. This score uses a Bayesian information criterion to penalise matches that are similar to the background distribution. We show that the BLiC score exhibits other, highly undesirable properties. Therefore as an alternative, we offer a general approach to adjust any motif similarity score so as to reduce the number of reported spurious alignments of uninformative columns. We implemented our method in Tomtom and we show that, without significantly compromising Tomtom's retrieval accuracy or runtime, we drastically reduce the number of uninformative alignments. The modified Tomtom is currently available as part of the MEME Suite at http://meme.nbcr.net. A motif is not limited to sites regulating gene expression. A motif is a recurring nucleotide sequence pattern that has a biological significance. One such example is in the context of the origins of replication of Saccharomyces cerevisiae. Autonomously replicating sequences (ARSs) are DNA fragments that promote extrachromosomal maintenance of plasmids. These ARSs mostly coincide with origins of DNA replication and therefore we use the terms interchangeably. The origins of replication in Saccharomyces cerevisiae have a highly conserved sequence known as the ACS (ARS consensus sequence). Depending on the reference, its representation varies from the 11bp consensus sequence WTTTAYRTTTW to a 33bp position weight matrix. While the replication origins of some species, such as Schizosaccharomyces pombe and metazoans, do not have any known motif, Liachko et al. (2010) found that the replication origins of another budding yeast Kluyveromyces lactis share a 50-bp ACS motif which is inherently different to the ACS motif found in S. cerevisiae. Here we characterise ARSs in Lachancea (Saccharomyces) kluyveri - a pre-whole genome duplication budding yeast. In addition, we demonstrate that ARS function in L. kluyveri is dependent on a much longer sequence compared with S. cerevisiae and K. lactis. Furthermore, the system of replication initiation in L. kluyveri appears to be more permissive than in these other two species - it is able to initiate replication from all S. cerevisiae ARSs and most K. lactis ARSs, while only half of L. kluyveri ARSs function in S. cerevisiae and less than 10% function in K. lactis. Our findings demonstrate a replication initiation system with novel features and underscore its functional diversity within the budding yeasts.
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CHALAOUX, FRANCOIS-REGIS. "Etude conformationnelle d'oligopeptides porteurs de la sequence consensus rxvrg, site d'action d'une endoprotease de la peau de xenopus laevis." Paris 6, 1994. http://www.theses.fr/1994PA066783.
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Le but de l'etude presentee dans ce document est de montrer l'apport de la determination de la structure tridimensionnelle en solution d'une serie d'oligopeptides lineaires contenant la sequence consensus rxvrg a la comprehension de leur maturation par l'endoprotease-rxvrg de la peau de xenopus laevis. En effet, les proteases impliquees dans les processus de maturation des precurseurs et porteurs de ces oligopeptides necessitent un site de coupure tres specifique. Cette specificite doit etre necessairement associee a l'existence de caracteristiques sequentielles et/ou conformationnelles permettant la reconnaissance entre l'endoprotease et son substrat. L'etude conformationnelle a ete realisee par #1h-rmn (incluant les spectres cosy, hohaha et noesy) sur un tetradecapeptide, mimant l'environnement du site de reconnaissance, ainsi que sur des derives substitues ou fragmentes afin de preciser le role des acides amines sur cette activite enzymatique et sur la structuration de la zone consensus. L'existence de structures repliees au niveau de la zone consensus du tetradecapeptide modele est mise en evidence par: (a) les valeurs negatives permises pour les angles dihedraux (autour de -20) de val#7 et arg#8 ; (b) l'existence de liaisons hydrogenes (ou du moins de protection du nh vis-a-vis du solvant) pour les residus 6, 7 et 8 ; (c) la presence des correlations n#7n#8 et n#8n#9 ; (d) l'observation des interactions a moyenne distance: arg#5hval#7nh et asp#6harg#8nh ; (e) le ralentissement des temps de correlation des residus de la sequence consensus ; (f) la modelisation moleculaire qui montre egalement une organisation structurales au niveau de la sequence consensus en utilisant les contraintes issues de l'etude rmn. Le remplacement du residu val#7 par un ser#7 dans la zone consensus rend le peptide moins sensible a la rxvrg-endoprotease et favorise l'action d'une autre metalloendoprotease (phie). Les resultats rmn sont en accord avec une structure etendue pour ce peptide. Le remplacement du residu ser#1#2 par un ala#1#2, en dehors de la sequence consensus, entraine une diminution de l'affinite de l'enzyme pour le substrat. La modelisation moleculaire de ce peptide montre la possibilite d'interpreter l'ensemble des donnees rmn avec une seule structure plus etendue que le tetradecapeptide modele au niveau de la sequence consensus. L'activite de la rxvrg endoprotease (coupure 8-9) est reduite de moitie avec le peptide (ala#1#2), tandis que l'organisation structurale est manifestement moins importante que dans le tetradecapeptide modele
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Nilsson, Johan. "Membrane protein topology : prediction, experimental mapping and genome-wide analysis /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7349-963-3/.
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DANCKAERT, PERRUCHOT ANNE. "Traitement informatique des autoradiographies de gels de sequences : conception et mise en oeuvre d'un systeme automatique d'interpretation d'autoradiographies, elaboration d'un algorithme de construction de la sequence consensus a partir de fragments." Paris 7, 1988. http://www.theses.fr/1988PA077045.
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MILHE, CATHERINE. "Flexibilite de l'adn : etude par rmn d'oligonucleotides en solution. structure et dynamique de points chauds de mutagenese et dynamique de la sequence consensus d'un promoteur." Université Louis Pasteur (Strasbourg) (1971-2008), 1994. http://www.theses.fr/1994STR13137.
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Ce travail porte sur l'etude structurale et dynamique, par resonance magnetique nucleaire (rmn), de sequences d'adn en solution. L'acetylaminofluorene (aaf) est un puissant cancerigene qui se fixe principalement en position c8 des guanines. Il induit, avec des frequences elevees, des mutations par deletion de 1 ou 2 paires de bases en des points chauds de mutagenese particuliers. La mise en place de la mutation pourrait intervenir soit par l'action d'un groupe d'enzymes qui reconnaitrait, sur l'adn endommage, une structure particuliere, soit au cours de la replication de l'adn avec formation d'intermediaires glisses. Afin de tester ces deux hypotheses, des etudes de rmn ont ete entreprises sur deux oligonucleotides mono-modifies par l'aaf, la modification de l'un n'entrainant pas de mutation, la modification de l'autre conduisant a un fort taux de mutagenese. Tres peu de differences tant structurales que dynamiques ont ete observees. D'autre part, l'etude par rmn d'intermediaires glisses a mis en evidence une forte stabilisation de l'intermediaire modifie fortement mutagene, laissant penser que le mecanisme de mutagenese est certainement replicatif. L'etude de la dynamique, par mesure de temps de relaxation dans le referentiel tournant, a montre qu'un fragment d'adn portant un site promoteur bacterien est soumis, a 25c, a un phenomene d'echange a deux sites dans la gamme de temps de la milliseconde. Cet equilibre a ete relie a l'hydratation de l'adn: a basse temperature, une gangue d'eau est fortement organisee autour de l'adn, alors qu'a haute temperature, elle a disparu. La temperature de 25c correspond a la temperature a laquelle l'eau d'hydratation se detache. L'equilibre conformationnel semble jouer un role biologique important: en dessous de 25c, la transcription ne peut pas demarrer
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Malaby, Heidi L. H. "Identification of Molecular Determinants that Shift Co- and Post-Translational N-Glycosylation Kinetics in Type I Transmembrane Peptides: A Dissertation." eScholarship@UMMS, 2014. https://escholarship.umassmed.edu/gsbs_diss/702.
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Asparagine (N)-linked glycosylation occurs on 90% of membrane and secretory proteins and drives folding and trafficking along the secretory pathway. The N-glycan can be attached to an N-X-T/S-Y (X,Y ≠ P) consensus site by one of two oligosaccharyltransferase (OST) STT3 enzymatic isoforms either during protein translation (co-translational) or after protein translation has completed (post-translational). While co-translational N-glycosylation is both rapid and efficient, post-translational N-glycosylation occurs on a much slower time scale and, due to competition with protein degradation and forward trafficking, could be detrimental to the success of a peptide heavily reliant on post-translational N-glycosylation. In evidence, mutations in K+ channel subunits that shift N-glycosylation kinetics have been directly linked to cardiac arrhythmias. My thesis work focuses on identifying primary sequence factors that affect the rate of N-glycosylation.
To identify the molecular determinants that dictate whether a consensus site acquires its initial N-glycan during or after protein synthesis, I used short (~ 100-170 aa) type I transmembrane peptides from the KCNE family (E1-E5) of K+ channel regulatory subunits. The lifetime of these small membrane proteins in the ER translocon is short, which places a significant time constraint on the co-translational N-glycosylation machinery and increases the resolution between co- and post-translational events. Using rapid metabolic pulse-chase experiments described in Chapter II, I identified several molecular determinants among native consensus sites in the KCNE family that favor co-translational N-glycosylation: threonine containing-consensus sites (NXT), multiple N-terminal consensus sites, and long C-termini. The kinetics could also be shifted towards post-translational N-glycosylation by converting to a serine containing-consensus site (NXS), reducing the number of consensus sites in the peptide, and shortening the C-termini.
In Chapter III, I utilized an E2 scaffold peptide to examine the N-glycosylation kinetics of the middle X residue in an NXS consensus site. I found that large hydrophobic and negatively charged residues hinder co-translational N-glycosylation, while polar, small hydrophobic, and positively charged residues had the highest N-glycosylation efficiencies. Poorly N-glycosylated NXS consensus sites with large hydrophobic and negatively charged X residues had a significantly improved co-translational N-glycosylation efficiency upon conversion to NXT sites.
Also in Chapter III, I adapted a siRNA knockdown strategy to definitively identify the OST STT3 isoforms that perform co- and post-translational N-glycosylation for type I transmembrane substrates. I found that the STT3A isoform predominantly performs co-translational N-glycosylation while the STT3B isoform predominantly performs post-translational N-glycosylation, in agreement with the roles of these enzymatic subunits on topologically different substrates.
Taken together, these findings further the ability to predict the success of a consensus site by primary sequence alone and will be helpful for the identification and characterization of N-glycosylation deficiency diseases.
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Malaby, Heidi L. H. "Identification of Molecular Determinants that Shift Co- and Post-Translational N-Glycosylation Kinetics in Type I Transmembrane Peptides: A Dissertation." eScholarship@UMMS, 2004. http://escholarship.umassmed.edu/gsbs_diss/702.
Texto completoResumen
Asparagine (N)-linked glycosylation occurs on 90% of membrane and secretory proteins and drives folding and trafficking along the secretory pathway. The N-glycan can be attached to an N-X-T/S-Y (X,Y ≠ P) consensus site by one of two oligosaccharyltransferase (OST) STT3 enzymatic isoforms either during protein translation (co-translational) or after protein translation has completed (post-translational). While co-translational N-glycosylation is both rapid and efficient, post-translational N-glycosylation occurs on a much slower time scale and, due to competition with protein degradation and forward trafficking, could be detrimental to the success of a peptide heavily reliant on post-translational N-glycosylation. In evidence, mutations in K+ channel subunits that shift N-glycosylation kinetics have been directly linked to cardiac arrhythmias. My thesis work focuses on identifying primary sequence factors that affect the rate of N-glycosylation.
To identify the molecular determinants that dictate whether a consensus site acquires its initial N-glycan during or after protein synthesis, I used short (~ 100-170 aa) type I transmembrane peptides from the KCNE family (E1-E5) of K+ channel regulatory subunits. The lifetime of these small membrane proteins in the ER translocon is short, which places a significant time constraint on the co-translational N-glycosylation machinery and increases the resolution between co- and post-translational events. Using rapid metabolic pulse-chase experiments described in Chapter II, I identified several molecular determinants among native consensus sites in the KCNE family that favor co-translational N-glycosylation: threonine containing-consensus sites (NXT), multiple N-terminal consensus sites, and long C-termini. The kinetics could also be shifted towards post-translational N-glycosylation by converting to a serine containing-consensus site (NXS), reducing the number of consensus sites in the peptide, and shortening the C-termini.
In Chapter III, I utilized an E2 scaffold peptide to examine the N-glycosylation kinetics of the middle X residue in an NXS consensus site. I found that large hydrophobic and negatively charged residues hinder co-translational N-glycosylation, while polar, small hydrophobic, and positively charged residues had the highest N-glycosylation efficiencies. Poorly N-glycosylated NXS consensus sites with large hydrophobic and negatively charged X residues had a significantly improved co-translational N-glycosylation efficiency upon conversion to NXT sites.
Also in Chapter III, I adapted a siRNA knockdown strategy to definitively identify the OST STT3 isoforms that perform co- and post-translational N-glycosylation for type I transmembrane substrates. I found that the STT3A isoform predominantly performs co-translational N-glycosylation while the STT3B isoform predominantly performs post-translational N-glycosylation, in agreement with the roles of these enzymatic subunits on topologically different substrates.
Taken together, these findings further the ability to predict the success of a consensus site by primary sequence alone and will be helpful for the identification and characterization of N-glycosylation deficiency diseases.
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Rajbhandari, Rajita [Verfasser], and Mathias [Akademischer Betreuer] Herrmann. "Studies on the role of the putative phosphorylation consensus sequence YXNX in the tandem repeat domains of the Staphylococcus aureus Mu50 Extracellular Adherence Protein (Eap) / Rajita Rajbhandari. Betreuer: Mathias Herrmann." Saarbrücken : Saarländische Universitäts- und Landesbibliothek, 2012. http://d-nb.info/1052222625/34.
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Vázquez-Figueroa, Eduardo. "Development of a novel dehydrogenase and a stable cofactor regeneration system." Diss., Georgia Institute of Technology, 2008. http://hdl.handle.net/1853/31685.
Texto completoResumen
The first goal of this work focused on the development of an amine dehydrogenase (AmDH) from a leucine dehydrogenase using site-directed mutagenesis. We aimed at reductively aminating a prochiral ketone to a chiral amine by using leucine dehydrogenase (LeuDH) as a starting template. This initial work was divided into two stages. The first focused mutagenesis to a specific residue (K68) that we know is key to developing the target functionality. Subsequently, mutagenesis focused on residues known to be in close proximity to a key region of the substrate (M65 and K68). This approach allowed for reduced library size while at the same time increased chances of generating alternate substrate specificity. An NAD+-dependent high throughput assay was optimized and will be discussed. The best variants showed specific activity in mU/mg range towards deaminating the target substrate.
The second goal of this work was the development of a thermostable glucose dehydrogenase (GDH) starting with the wild-type gene from Bacillus subtilis. GDH is able to carry out the regeneration of both NADH and NADPH cofactors using glucose as a substrate. We applied the structure-guided consensus method to identify 24 mutations that were introduced using overlap extension. 11 of the tested variants had increased thermal stability, and when combined a GDH variant with a half-life ~3.5 days at 65℃ was generated--a ~10⁶increase in stability when compared to the wild-type.
The final goal of this work was the characterization of GDH in homogeneous organic-aqueous solvent systems and salt solutions. Engineered GDH variants showed increased stability in all salts and organic solvents tested. Thermal stability had a positive correlation with organic solvent and salt stability. This allowed the demonstration that consensus-based methods can be used towards engineering enzyme stability in uncommon media. This is of significant value since protein deactivation in salts and organic solvents is not well understood, making a priori design of protein stability in these environments difficult.
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Vargas, Jesse. "Protein-DNA interactions : characterization of a definitive DNA binding sequence consensus for full length mammalian DEAF-1 and determination of the DNA binding potential of the tumor metastasis related protein, MTA1 /." Available to subscribers only, 2007. http://proquest.umi.com/pqdweb?did=1483331711&sid=2&Fmt=2&clientId=1509&RQT=309&VName=PQD.
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Liu, Chong. "EVIDENCE FOR THE MATURATION OF CELLULAR IMMUNE RESPONSES IN EQUINE INFECTIOUS ANEMIA VIRUS-INFECTED PONIES." UKnowledge, 2013. http://uknowledge.uky.edu/gluck_etds/10.
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Equine infectious anemia virus (EIAV) has been used as a model to investigate protective mechanisms against lentiviruses. Unlike other lentiviruses, EIAV replication can be eventually controlled in most infected horses leading to an inapparent carrier state free of overt clinical signs which can last for many years. Maintenance of this carrier state is absolutely dependent on active immune responses as evidenced by the fact that immunosuppressive drugs can induce the recurrence of disease. However, the immune mechanisms that are responsible for this control of infection are not yet identified. As the resolution of the initial infection is correlated with the appearance of the virus-specific cytotoxic T lymphocytes (CTL), it appears that cellular immune responses play an important role. However, most studies into this protective mechanism have been limited to the identification of specific epitopes, usually at a single time point in the infection. Few studies have examined the cellular immune responses to the viral antigens throughout the infection period. Since the virus undergoes rapid mutation following infection, the adaptive immune response must also evolve to meet this challenge. Previously, the EIAV envelope (gp90) protein was shown to be the primary determinant of vaccine efficacy. Here, we hypothesized that the maturation of cellular immune responses is a lengthy process and involves envelope-specific T cell recognition shifting from immunodominant variable determinants to conserved immunorecessive determinants during the initial stages of the EIAV infection. The first part of this dissertation was to develop a new in vivo method to identify envelope-specific T cell responses. The second part of this dissertation was to investigate whether envelope-specific T cell recognition evolved in EIAV-infected ponies. Finally, the mechanisms for this T cell immunodominant shifting were also investigated from the point of both virus sequence mutation and T cell clone expansion and contraction. Also, a new EIAV attenuated vaccine which contained a consensus gp90 sequence was tested to see if it facilitated T cell recognition of the more conserved regions early in the infection. Our results indicated that envelope-specific T cell recognition patterns changed over time. Early after infection, dominant immune responses to the peptides in the carboxyl-terminus variable region were identified. By six months post infection, the recognized peptides spanned the entire envelope sequence, with a shift to the amino-terminus conserved region. The mechanisms responsible for this change remain unclear, but analysis of T cell receptor repertoire indicated that T cell clonal expansion and contraction might be one of the reasons. Our demonstration that envelope-specific peptide recognition shifts from the variable to the more conserved regions provides evidence that the maturation of cell mediated immune response is parallelled with long-term control of this infection.
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Kao, Daniel Joseph. "Development of a synthetic peptide vaccine and antibody therapeutic for the prevention and treatment of Pseudomonas Aeruginosa infection /." Connect to full text via ProQuest. Limited to UCD Anschutz Medical Campus, 2007.
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Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2007.<br>Typescript. Includes bibliographical references (leaves 203-212; 260-261). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;
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Bellora, Pereyra Nicolás. "In silico analysis of regulatory motifs in gene promoters." Doctoral thesis, Universitat Pompeu Fabra, 2010. http://hdl.handle.net/10803/7202.
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Regulation of gene transcription is a complex process involving many different proteins, some of which bind in a sequence-specific manner to DNA motifs in the gene promoter. The need to maintain specific interactions between transcription factors and proteins involved in the RNA polymerase II complex is expected to impose constrains on the relative position and spacing of the interacting DNA motifs. The present work includes the development of a novel approach to identify motifs that show a preferential location in DNA sequences and the implementation of a public web application called PEAKS. We investigated if the arrangement and nature of the most common motifs depended on the breath of expression of the gene. We found differences that serve to illustrate that many key specific regulatory signals may be present in the proximal promoter region in mammalian genes. We also apply other methods for the identification of specific transcription factors (TFs) involved in the co-regulation of a set of genes. Data from experimentally-verified transcription factors binding sites (TFBSs) support the biological relevance of our findings.<br>La regulació de la transcripció dels gens és un procés complex que implica moltes proteïnes diferents, algunes de les quals s'unexien a motius específics d'ADN localitzats a la regió promotora dels gens. S'espera que la necessitat de mantenir les interaccions específiques entre els factors de transcripció i les proteïnes implicades en el complex de la ARN polimerasa II imposi limitacions en la posició relativa i l'espaiat dels motius d'interacció amb l'ADN. La feina presentada en aquesta tesi inclou el desenvolupament d'un nou metode per l'identificació de motius que mostren una localització preferencial en seqüències d'ADN i l'implementació d'una aplicació web pública anomenada PEAKS. Hem investigat si la col·locació i la naturalesa de la majoria dels motius comuns depen del rang d'expresió del gen. Hem trobat diferències que serveixen per il·lustrar el fet que moltes senyals clau de regulació gènica poden estar presents en la regió proximal del promotor dels gens de mamífers. També hem aplicat altres mètodes per a l'identificació de factors de transcripció (TFs) específics involucrats en la co-regulació d'un grup de gens. Dades de llocs d'unio dels TFs (TFBSs) verificats experimentalment recolzen la rellevància biològica dels nostres resultats.
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Mlera, Luwanika. "Preparatory investigations for developing a transcript-based rotavirus reverse genetics system / Luwanika Mlera." Thesis, North-West University, 2012. http://hdl.handle.net/10394/9523.
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Reverse genetics systems that are based on either viral transcripts or cDNA genome
segments cloned in plasmids have recently been reported for some of the dsRNA
viruses of the Reoviridae family, namely African horsesickness virus, bluetongue
virus and orthoreovirus. For rotaviruses, three reverse genetics systems which only
allow the manipulation of a single genome segment have been described. These
rotavirus single genome segment reverse genetics systems are not true stand-alone
systems because they require a helper virus and a recombinant virus selection step.
A true selection-free, plasmid- only or transcript-based reverse genetics system for
rotaviruses is lacking.
This study sought to identify and characterise the factors that need to be understood
and overcome for the development of a rotavirus reverse genetics system using
mRNA derived from the in vitro transcription of a consensus nucleotide sequence as
well as from double-layered particles. The consensus whole genome sequence of
the prototype rotavirus DS-1 and SA11 strains was determined using sequenceindependent
whole genome amplification and 454® pyrosequencing. For the
rotavirus DS-1 strain, a novel isoleucine in a minor population variant was found at
position 397 in a hydrophobic region of VP4. NSP1 contained seven additional
amino acids MKSLVEA at the N-terminal end due to an insertion in the consensus
nucleotide sequence of genome segment 5. The first 34 nucleotides at the 5'-
terminus and last 30 nucleotides at the 3'-terminal end of genome segment 10
(NSP4) of the DS-1 strain were determined in this study. The consensus genome
segment 11 (NSP5/6) sequence was 821 bp in length, 148 bp longer than previously
reported. The 454® pyrosequence data for a rotavirus SA11 sample with no known
passage history revealed a mixed infection with two SA11 strains. One of the strains
was a reassortant which contained genome segment 8 (NSP2) from the bovine
rotavirus O agent. The other ten consensus genome segments of the two strains
could not be differentiated. Novel minor population variants of genome segments 4
(VP4), 9 (VP7) and 10 (NSP4) were identified. Molecular clock phylogenetic
analyses of the rotavirus SA11 genomes showed that the two SA11 strains were
closely related to the original SA11-H96 strain isolated in 1958. Plasmids containing inserts of the consensus cDNA of the rotavirus DS-1 strain were
purchased and used to generate exact capped transcripts by in vitro transcription
with a T7 polymerase. Wild-type transcripts of rotavirus SA11 were obtained from in
vitro transcription using purified rotavirus SA11 double-layered particles. The purified
rotavirus DS-1 and SA11 transcripts were transfected into BSR, COS-7 and MA104
cells. Work on MA104 cells was discontinued due their very low transfection efficacy.
In BSR and COS-7 cells, rotavirus DS-1 and SA11 transcripts induced cell death.
However, no viable rotavirus was recovered following attempts to infect MA104 cells
with the BSR and COS-7 transfected cell lysates. The cell death was determined to
be due to apoptotic cell death mechanisms. Immunostaining showed that the DS-1
genome segment 6 (VP6) and SA11 transcripts were translated in transfected BSR
and COS-7 cells. Based on visual inspection, the translation seemed to be higher in
the retinoic acid-inducible gene-I (RIG-I) deficient BSR cells than in COS-7 cells.
This suggested that the transfection of rotavirus transcripts induced an innate
immune response which could lead to the development of an antiviral state.
Therefore, the innate immune response to rotavirus transcripts was investigated in
HEK 293H cells using qRT-PCR and western blot analyses. Results of this
investigation showed that RIG-I, but not MDA5 sensed rotavirus transcripts in
transfected HEK 293H cells. Furthermore, rotavirus transcripts induced high levels of
cellular mRNA encoding the cytokines IFN-1β, IFN-λ1, CXCL10 and TNF-α. Other
cytokines namely, IFN-α, IL-10, IL-12 p40 and the kinase RIP1 were not significantly
induced. Inhibiting the RNA-dependent protein kinase R (PKR) reduced the induction
of cytokines IFN-1β, IFN-λ1, CXCL10 and TNF-α, but the expression levels were not
abrogated. The importance of a consensus sequence and the insights gained in the
current study regarding the role of the innate immune response after transfection of
rotavirus transcripts into cells in culture, should aid the development of a true
rotavirus reverse genetics system.<br>Thesis (PhD (Biochemistry))--North-West University, Potchefstroom Campus, 2013
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Liang, Chengzhi. "COPIA: A New Software for Finding Consensus Patterns in Unaligned Protein Sequences." Thesis, University of Waterloo, 2001. http://hdl.handle.net/10012/1050.
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Consensus pattern problem (CPP) aims at finding conserved regions, or motifs, in unaligned sequences. This problem is NP-hard under various scoring schemes. To solve this problem for protein sequences more efficiently,a new scoring scheme and a randomized algorithm based on substitution matrix are proposed here. Any practical solutions to a bioinformatics problem must observe twoprinciples: (1) the problem that it solves accurately describes the real problem; in CPP, this requires the scoring scheme be able to distinguisha real motif from background; (2) it provides an efficient algorithmto solve the mathematical problem. A key question in protein motif-finding is how to determine the motif length. One problem in EM algorithms to solve CPP is how to find good startingpoints to reach the global optimum. These two questions were both well addressed under this scoring scheme,which made the randomized algorithm both fast and accurate in practice. A software, COPIA (COnsensus Pattern Identification and Analysis),has been developed implementing this algorithm. Experiments using sequences from the von Willebrand factor (vWF)familyshowed that it worked well on finding multiple motifs and repeats. COPIA's ability to find repeats makes it also useful in illustrating the internal structures of multidomain proteins. Comparative studies using several groups of protein sequences demonstrated that COPIA performed better than the commonly used motif-finding programs.
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Almeida, Rafael Ribeiro. "Reconhecimento entre clados e efeito supressor induzido por vacinas de DNA codificando peptídeos conservados e promíscuos do grupo M do HIV-1." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/5/5146/tde-29102014-160344/.
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A busca por uma construção vacinal contra o HIV-1 é urgente. Os linfócitos T CD4+ têm assumido um papel de destaque no campo de vacinas por participar no controle da replicação do HIV-1, seja auxiliando as funções efetoras de linfócitos T CD8+ e a produção de anticorpos por linfócitos B ou mesmo agindo de forma citotóxica sobre macrófagos infectados. A utilização de sequências consenso do grupo M do HIV-1 é apontada como uma das maneiras de se contornar os problemas relacionados à diversidade viral. Além disso, é preciso construir vacinas que apresentem potencial de induzir respostas imunes com grande cobertura populacional. Com o intuito de induzir respostas amplas de linfócitos T CD4+ contra diversos subtipos do HIV-1 em uma população geneticamente diversa para moléculas HLA-DR, identificamos em nosso trabalho prévio 34 peptídeos promíscuos (previstos de se ligarem a múltiplas moléculas HLA-DR) e conservados da sequência consenso do grupo M do HIV-1. Desenvolvemos uma vacina de DNA codificando 7 peptídeos de Env (HIVenv7) e outra vacina (HIVBr27) codificando os demais 27 peptídeos. A vacina HIVBr27 foi imunogênica em camundongos BALB/c, induzindo uma resposta ampla e polifuncional de linfócitos T CD4+ e CD8+. A vacina HIVenv7 foi pouco imunogênica e mostrou-se capaz de suprimir a resposta induzida pela HIVBr27 em regime de co-imunização. No presente trabalho demonstramos que a imunização com HIVBr27 induz uma resposta imune celular mediada por linfócitos T CD4+ e CD8+ contra peptídeos de diferentes subtipos do HIV-1. Além disso, a imunização com HIVBr27 mostrou-se parcialmente protetora contra a infecção pelo vírus Vaccinia recombinante codificando as proteínas Gag e Pol do HIV-1. Ensaios in vitro demonstraram que os peptídeos codificados pela HIVBr27 se ligam a múltiplas moléculas HLA de classe II e são reconhecidos por células de pacientes infectados pelo HIV-1. Demonstramos também que a vacina HIVenv7 não possui propriedades imunossupressoras consistentes, contrariando os resultados obtidos previamente. Os peptídeos codificados pela HIVenv7 se ligaram a múltiplas moléculas HLA de classe II, mas apresentaram baixa frequência de reconhecimento por células de pacientes infectados pelo HIV-1. Acreditamos que a vacina HIVBr27 possui potencial de induzir uma resposta imune de grande cobertura populacional e direcionada a diferentes variantes do HIV-1. Por outro lado, a vacina HIVenv7 se mostrou pouco imunogênica e não deve ser utilizada em estudos futuros<br>The search for an HIV-1 vaccine construct is urgent. The CD4+ T cells have assumed a prominent role in the vaccine field participating in the control of HIV-1 replication either by helping CD8+ T cell effector function and B cell-mediated antibody production or by acting as citotoxic cells on infected macrophages. The use of HIV-1 M-group consensus sequences is pointed as an alternative to overcome viral diversity. Besides, it is necessary to construct vaccines that would potentially induce immune responses with broad population coverage. Intending to induce a broad CD4+ T-cell immune response against different HIV-1 subtypes in a population bearing diverse HLA-DR molecules we have previously identified 34 promiscuous peptides (potentially binding to multiple HLA-DR molecules) and conserved within the HIV-1 M-group consensus sequence. We construct a DNA vaccine encoding 7 Env peptides (HIVenv7) and another vaccine (HIVBr27) encoding 27 peptides. The HIVBr27 vaccine was immunogenic in BALB/c mice, inducing a broad and polyfunctional CD4+ and CD8+ T-cell response. The HIVenv7 vaccine was much less immunogenic and suppressed HIVBr27-induced immune responses when co-immunized. Here, we have shown that HIVBr27 immunization leads to a cross-clade CD4+ and CD8+ T-cell immune response. Besides, HIVBr27 immunization has partially protected mice challenged with a recombinant Vaccinia virus encoding HIV-1 Gag e Pol. In vitro assays have shown that HIVBr27- encoded peptides bind to multiple HLA class II molecules and are recognized by HIV- 1-infected patients. We have also shown that HIVenv7 has no consistent immunosuppressive properties, contradicting our previous results. The HIVenv7- encoded peptides bound to multiple HLA class II molecules but were recognized by a low number of HIV-1-infected patients. We believe that our vaccine HIVBr27 has potential to induce an immune response with broad population coverage, towards different HIV-1 variants. On the other hand, the HIVenv7 vaccine was poorly immunogenic and should not be used in future studies
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Clarke, Andrew, Stefan Prost, Jo-Ann Stanton, W. T. White, Matthew Kaplan, Elizabeth Matisoo-Smith, and Genographic Consortium The. "From cheek swabs to consensus sequences: an A to Z protocol for high-throughput DNA sequencing of complete human mitochondrial genomes." BioMed Central, 2014. http://hdl.handle.net/10150/610024.
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BACKGROUND:Next-generation DNA sequencing (NGS) technologies have made huge impacts in many fields of biological research, but especially in evolutionary biology. One area where NGS has shown potential is for high-throughput sequencing of complete mtDNA genomes (of humans and other animals). Despite the increasing use of NGS technologies and a better appreciation of their importance in answering biological questions, there remain significant obstacles to the successful implementation of NGS-based projects, especially for new users.RESULTS:Here we present an 'A to Z' protocol for obtaining complete human mitochondrial (mtDNA) genomes - from DNA extraction to consensus sequence. Although designed for use on humans, this protocol could also be used to sequence small, organellar genomes from other species, and also nuclear loci. This protocol includes DNA extraction, PCR amplification, fragmentation of PCR products, barcoding of fragments, sequencing using the 454 GS FLX platform, and a complete bioinformatics pipeline (primer removal, reference-based mapping, output of coverage plots and SNP calling).CONCLUSIONS:All steps in this protocol are designed to be straightforward to implement, especially for researchers who are undertaking next-generation sequencing for the first time. The molecular steps are scalable to large numbers (hundreds) of individuals and all steps post-DNA extraction can be carried out in 96-well plate format. Also, the protocol has been assembled so that individual 'modules' can be swapped out to suit available resources.
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Moore, Siobhan E. "Fertility practitioners' experience of the psychological sequelae of unmet parental goals after unsuccessful fertility treatment : a Delphi Consensus Study." Thesis, Cardiff University, 2018. http://orca.cf.ac.uk/115247/.
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This thesis investigates the adjustment processes to involuntary childlessness and the psychological distress associated with unmet parental goals. Paper one reports a systematic review of quantitative studies looking at how trauma theory informs the clinical understanding of adjustment to involuntary childlessness. This yielded eight studies which were reviewed and rated using a quality appraisal tool. The data extracted, focused on the prevalence of trauma and post traumatic growth to infertility. No studies included, focused their research on cohorts of women who identified as being involuntarily childless through delayed childbearing or circumstantial reasons. The findings suggested that for women who are, infertile, who had accessed or were accessing fertility treatment and were childless, trauma theory can aid clinical understanding of both their experience of infertility distress and adjustment to involuntary childlessness. Paper two describes a three round, online Delphi Study which investigated, infertility practitioners’ clinical experience of psychological distress associated with unmet parental goals, following unsuccessful fertility treatment. Nine practitioners, from five countries participated, rating 58 statements on the presentation and nature of distress observed in the post treatment phase. Infertility practitioners perceived distress to be associated with statements concerned with individual’s identity and relinquishing the desire for biological children. The fertility practitioners agreed that the core element of therapy was to facilitate meaning making, acceptance and pursuit of new life goals. Paper three provides a critical account of the strengths and limitations of both the systematic review and empirical paper. The theoretical and clinical implications of the research included addressing pertinent issues, which arose during the research process. Finally, the competencies developed from conducting this research will be described in relation to becoming a clinical psychologist.
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Chu, Huan-Lin, and 朱煥霖. "Mining Consensus Patterns Across Heterogeneous Sequence Databases." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/zf9kav.
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碩士<br>國立中正大學<br>資訊管理系研究所<br>104<br>Many modern enterprise collect large quantities of data, such as customer preference or their temporal purchasing behaviors, and utilize data mining as their competitive advantages. However, some conflicts in the data may exist, and determining how to aggregate many different opinions into a consensus is a traditional core problem in recommendation systems and decision support systems. Taking the preference ranking problem as an example, u_1 : (A ≥ B ≻ C) indicates that for the user u_1, (1)A is at least favorable than B; (2)B is more favorable compared to C. Another temporal ranking problem is to discover the possible temporal relationships among items, which refer to the temporal ordering of items. For example, u_1 : (A < B = C) indicates that the user considers that item A should occur before B and item B can occur simultaneously with C. However, in the real world, user may have many different aspects of consideration in regards to the same itemset at the same time. The real-life application is that when investors purchase stocks, they consider not only the stock preference due to personal risk tolerance, but also the temporal order of stock investment to maximize the profit because of the temporal effects of supply chain positions. Based on the above ideas, this study defines a novel model and proposes its associated algorithm for discovering consensus patterns combining preference ranking and temporal sequence. A two-phase experiment was designed to collect authentic datasets, execute the algorithm for its effectiveness via user rating, and demonstrate its managerial meaning.
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劉玉山. "Using Tabu Search for Finding Consensus Sequence." Thesis, 2004. http://ndltd.ncl.edu.tw/handle/07094808649625506348.
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碩士<br>樹德科技大學<br>資訊管理研究所<br>92<br>A consensus sequence captures the common features of a set of sequences. Consensus sequences are important in various DNA sequencing applications and are a convenient way to characterize a family of molecules. The search of such a sequence is an NP-Complete problem and, therefore, no efficient algorithms to search the sequence can be designed. Tabu Search has been used in a wide variety of applications to find solutions for NP-Complete problems. In this paper, we use a variant of Tabu search to find a consensus sequence. The method may be viewed as consisting of two alternating phases, an Improving Phase and a Mixed Phase. And the resulting consensus sequences can be used to induce a multiple sequence alignment. The proposed approach has been experimented on real sequences and simulated sequences. The experimental results are compared with ClustalW to illustrate the effectiveness of the proposed approach.
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Chang, Yi-Feng, and 張益峰. "Human Promoter Prediction with Extracted Consensus Sequence Patterns." Thesis, 2003. http://ndltd.ncl.edu.tw/handle/73014006863284314043.
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碩士<br>朝陽科技大學<br>資訊管理系碩士班<br>91<br>Promoter region is a DNA sequence that is usually located on the upstream of the transcriptional starting site (TSS) of a gene. If the hallmarks of the known promoter sequences can be extracted, we can use these hallmarks to recognize unknown promoter regions from un-labeled genome sequences. Furthermore, we can indirectly identify the potential TSSs of genes, and then the estimated thirty to fifty thousand of genes can be predicted and explored.
Up to present, there are many announced promoter sequences that were discovered by molecular biologists with biological experiments. However, the process of discovering promoter sequences with traditional lab experiments is very time-consuming and costly. Therefore, many researchers take the advantage of high throughput analysis of bioinformatics for predicting promoter sequences. But, while those promoter prediction tools deal with the unknown and complex DNA sequences, they result in either low true positive or high false positive. For the above reasons, present promoter prediction tools still cannot be the consulting basis for genes identification.
Hence, in this thesis we first downloaded human promoter and non-promoter sequences from NCBI GenBank. After careful filtering, these promoter sequences were saved into our own designed promoter database called Promoter Databank. Next, a genetic algorithm based consensus sequence extracting program derived the promoter-specific and non-promoter-specific consensus sequence patterns from the promoter and non-promoter sequences. By applying a weighted-sum approach with the extracted consensus sequence patterns as detecting signals, we can predict if any unknown DNA sequence contains promoter sequences or not.
However, for the weight-sum approach, there is no way that a correct weight can be found and assigned for every set of consensus sequence patterns. Besides, the weighted-sum approach is based on string matching, and thus it makes the weighted-sum approach become time consuming and unsuitable for on-line prediction. For these reasons, this thesis proposed another two-phase neural network promoter prediction tool to improve the prediction accuracy and time complexity.
From the experiment results we found that, compared to the other existing promoter prediction tools, our proposed promoter prediction tools have better true positive and lower false positive rates. We believe this is because genetic algorithms can extract a large amount of uniformly distributed promoter-specific and non-promoter-specific consensus sequence patterns; and more sequence patterns lead to better prediction accuracy. Furthermore, because of the inclusion of non-promoter sequences, our proposed tools can also reduce the false positive rates.
In the future, the research idea and methods proposed in this thesis can be further applied to compare different organisms’ promoter sequences by adding other types of DNA sequences, such as repetitive sequences or intron sequences.
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Chan, Viann Wendy. "Comparative sequence analysis : prediction of consensus Xist RNA secondary structure." Thesis, 2003. http://hdl.handle.net/2429/14473.
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The typical female mammal has two X chromosomes. However, only one copy remains active, while the other copy is silenced. The Xist gene has been found to remain active on the silenced X chromosome. Other lab experiments have determined that the Xist RNA plays a significant role in the process of X - chromosome inactivation. Our goal in this thesis is to predict a possible consensus structure for the Xist RNA using comparative sequence analysis. Since the Xist RNA structure is not known, we evaluate our approaches on sequences with known structures. There are three main phases for this approach: (i) aligning multiple sequences, (ii) finding stems, and (iii) building the structure. Phases (ii) and (iii) are the main focus of this research. We used the concept of mutual information content to detect potential stems containing mutations. We found potential conserved stems by searching for sequences of complementary paired bases. The stems were then used to find the consensus structure. We investigated two general approaches for building the consensus structure, greedy and randomized methods. We tested these methods on data comprising the following types of RNA: tRNA, 16S rRNA, 23S rRNA, and Xist RNA. We found that our methods accurately predicted the consensus structure for tRNA. However, when we analyzed data sets with longer sequences, the accuracy of our predictions decreased. Of the methods we tested, we found that one of our greedy approaches performed better than the others on all the data sets. We predicted several possible structures for Xist from different alignments. While the overall accuracy of our predicted structures is likely to be low, some of the substructures predicted may give us clues about the true consensus structure.
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黃自健. "Identification Of A Consensus Sequence Motif For Autophosphorylation-Dependent Protein Kinase." Thesis, 1994. http://ndltd.ncl.edu.tw/handle/30520107396656820090.
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碩士<br>國立清華大學<br>生物醫學研究所<br>82<br>In a previous report (Yang et al., (1987a) J. Biol. Chem. 262, 7034 - 7040), a cyclic AMP and calcium - independent brain kinase which requires autophosphorylation for activity was identified as a very potent myelin basic protein (MBP) kinase. In this report, the phosphorylation sites of MBP by this autophosphorylation - dependent protein kinase (auto - kinase) were further determined by two - dimensional electrophoresis / thin - layer chromatography, phosphoamino acid analysis, high performance liquid chromatography, tryptic peptide mapping, sequential manual Edman degradation and direct peptide sequencing. Auto - kinase phosphorylates MBP on both threonine and serine residues. Three major tryptic phosphopeptide peaks were resolved by C18 - reversed phase high pefformance liquid chromatography. Sequential manual Edman degradation together with direct sequence analysis revealed that FS (p) WGAEGQKPGFGYGGR is the phosphorylation site sequence (molar ratio ~ 1.0) for the first major phosphopeptide peak. When mapping with bovine brain MBP sequence, we finally demonstrate Ser115, one of the in vivo phosphorylation sites in MBP, as the major site phosphorylated by auto - kinase, implicating a physiologically relevant role of auto - kinase in the regulation of brain myelin function. By using the same approach, we also identified HRDT (p) GILDSLGR (molar ratio ~ 0.9) and TT (p) HYGSLPQK (molar ratio ~ 0.8) as the major phosphorylation sites sequences in 32p - MBP phosphorylated by auto - kinase, further indicating that - Arg - X - Ser / Thr - (neutral amino acid) 3 - (amino acid containing hydroxyl group such as Ser / Glu / Asp) - (neutral amino acid) 2 - may represent a unique consensus sequence motif specifically recognized by this autophosphorylationdependent multisubstrate / multifunctional protein kinase in the brain.
To further confirm the consensus sequence motif for this autophosphorylation-dependent protein serine / threonine kinase, we have tested several synthetic peptides. The well - established protein serine / threonine kinases such as cAMP - dependent protein kinase (PKA), Ca2 + / calmodulin - dependent protein kinase (CaM - kinase) and protein kinase C (PKC) were found to be inactive towards phosphorylation of synitde - 3 (RPRPASVPPS - PSLSRHA), which turned out to be an excellent substrate only for auto - kinase, indicating that syntide - 3 is a specific substrate for auto - kinase, indicating that syntide - 3 is a specific substrate for auto - kinase. Modification of syntide - 3 to become RPRPASVPPS / T did not affect the activity of auto - kinase. By contrast, auto - kinase. rather or almost inactive when the peptide was modified to become RPRPASVPPA / G / F / K / R / D / E / Y, indicating that amino acid number 10 in syntide - 3 is crucial to the sequence motif recognized by auto - kinase. Taken together, the results provide initial evidence that - Arg - X - (X) - Ser / Thr - X3 - (amino acid containing hydroxyl group) - may represent a unique consensus sequence motif specifically recognized by autophosphorylation - dependent protein kinase, a new family of multisubstrate / multifunctional protein serine / threonine kinase.
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Kolychev, Dmitri. "Microsatellite detection and consensus sequence verification by virtual PCR and machine learning." 2003. http://purl.galileo.usg.edu/uga%5Fetd/kolychev%5Fdmitri%5Fs%5F200308%5Fms.
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Woodworth, AM. "Gene regulation by RUNX1 in the absence of consensus sequences." Thesis, 2020. https://eprints.utas.edu.au/35253/1/Woodworth_whole_thesis.pdf.
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Runt-related transcription factor 1 (RUNX1) is a transcription factor that has an important role in haematopoietic cell development and function and is frequently disrupted in leukaemia. RUNX1 is commonly described as a sequence-specific DNA binding factor which recognises the consensus sequence TG(T/C)GGT in the promoter and enhancer regions of its target genes to affect changes in gene expression. However, the advent of techniques to study DNA-protein interactions on a genome-wide scale has provided the opportunity to re-assess RUNX1 localisation and function, and this analysis suggests that the classical model of RUNX1 function is incomplete. By fully understanding the mechanisms by which RUNX1 maintains its target gene expression profiles under normal cellular conditions, insights into disrupted function can be gained and interventions can be developed.
Analysis of publicly available RUNX1 ChIP-Seq data determined that the majority of RUNX1 binding in haematopoietic cells occurs outside of gene promoter regions, in intergenic or intronic regions. Furthermore, approximately one fifth of all RUNX1-DNA binding sites on a genome-wide scale were not associated with a canonical consensus sequence, and this was particularly prevalent in promoter regions, with almost half of RUNX1 binding in promoter regions occurring in the absence of consensus sequences. This suggests that recruitment of RUNX1 to gene targets occurs through multiple mechanisms and raises the possibility that it may function differently depending on its location and mode of recruitment. Similar results were obtained for localisation of the RUNX1 fusion protein RUNX1-ETO, the product of a common chromosomal translocation in leukaemia.
This data set was used to investigate the different modes of binding and action of RUNX1 with the aim of establishing whether binding in the absence of consensus sequences constitutes a novel mechanism of RUNX1 binding and if genes regulated in this way respond differently to RUNX1 disruption. This study identified biological pathways enriched for genes with RUNX1 binding in their promoters, and in which only one type of binding (in the presence or absence of a consensus sequence) occurred. Clusters of functionally related RUNX1-bound genes were selected from two of these pathways; the RhoA and HMGB1 signalling pathways and used as models to investigate regulation by RUNX1.
The RhoA signalling pathway genes, ARHGAP1, ARHGAP4 and ARHGAP12, all bound RUNX1 at their promoters, in association with a consensus sequence. However, while ARHGAP4 and ARHGAP12 responded to RUNX1 in reporter assays, ARHGAP1 did not, suggesting that a consensus sequence alone is not sufficient for a promoter to respond to RUNX1. In contrast, a group of histone acetyl transferase genes from the HMGB1 signalling pathway bound RUNX1 at their promoters in the absence of a consensus sequence. Both the KAT6B and KAT2B promoters were activated by RUNX1 in the absence of a consensus sequence, suggesting a mechanism whereby RUNX1 recruitment to DNA does not require the canonical consequence sequence and may rely on recruitment by additional transcription factors or distal chromatin elements. Interestingly, evidence presented here indicates that while RUNX1-ETO inhibits RUNX1 activity at classically regulated promoters which contain RUNX consensus sequences, it cannot inhibit RUNX1 activity in the absence of canonical consensus sequences. This suggests that genes regulated by RUNX1 through different transcriptional mechanisms may respond differently to disruption of RUNX1 in diseases such as leukaemia.
The majority of RUNX1 binding occurs outside of promoters, with one potential explanation for intergenic or intronic RUNX1 binding being that it functions at gene enhancers. Data presented here identified two such potential enhancers of the HAT1 gene. While HAT1 bound RUNX1 at its promoter, although in the absence of a consensus sequence, it does not respond directly to RUNX1 in reporter assays. Potential enhancers were identified approximately 31 kb upstream and 190 kb downstream of the promoter. While interactions with these regions were confirmed, neither were found to behave as an enhancer in reporter assays. However, the data presented is consistent with the +190 kb region representing a promoter-promoter interaction which may be related to the formation of large interconnected transcriptional complexes. Such promoter-promoter interactions could at least partly explain the prevalence of RUNX1 binding at promoters in the absence of a consensus sequence.
This study has expanded our understanding of the mechanisms by which RUNX1 affects transcription of its target genes and has characterised distinct modes of operation at candidate genes: either directly through a consensus sequence; directly in the absence of a consensus sequence; or distally through an interacting regulatory element.
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Sung, Wen-Long, and 孫文隆. "Identification of the consensus sequence of the SOS box in Xanthomonas axonopodis pv. citri." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/58471540096145062408.
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碩士<br>輔仁大學<br>生命科學系碩士班<br>93<br>There are two lexA genes in Xanthomonas axonopodis pv. citri. LexA1 and LexA2 proteins, which are supposed to function as a repressor and modulate many DNA repair genes. By analyzing two sequences of LexA proteins, we discover they are not the same as the relative sequences (α domain) that bind to DNA. Therefore, we assume that the two proteins bind to different DNA sequences. In order to get further evidence, Electrophoretic mobility shift assay (EMSA) is adopted. The result shows that LexA1 protein can bind to lexA1 promoter (nucleotides -40 to +155, relative to the translation start site, +1),rather than to lexA2 promoter (nucleotides -71 to +134, relative to the translation start site, +1); on the contrast, LexA2 protein functions reversely, only bind to lexA2 promoter. LexA1 protein functions on where lexA1 promoter locates, which is known as an inverted repeat, 5’-TTAGTAGTAATACTACTAA-3’. In order to know where LexA2 protein functions, we analyze the shorten sequence of lexA2 promoter with LexA2 protein by EMSA. The result indicates that LexA2 protein binds to another sequence, 5’-GGTGTACAAATGTACACC-3’, which is different from the original one (TTAG-N8~11-CTAA). To get a better understanding of the symmetry of the sequence and the necessity of nucleotides in the sequence, EMSA is performed to determine the function of mutated sequence with the LexA2 protein. The data shows that the necessary sequence to which LexA2 protein binds probably is TGTN8~10ACA. We discover that, at the place where 115 bp upstream of the translation start site of the lexA2 gene, a specific sequence (5’-TTAGTACTAAAGTTATAA-3’), which is similar to the sequence (TTAG-N8~11-CTAA), to which LexA1 protein binds. We find that LexA1 protein can bind to this specific sequence, but the affinity is not as well as to the LexA1 protein on lexA1 promoter. Besides LexA1 protein can function on recA promoter, EMSA analysis is conducted to know if it works with LexA2. The result demonstrates LexA2 protein can also function on recA promoter.
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Cheng, Li-chen, and 鄭麗珍. "Mining maximum consensus sequences from group ranking data." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/58303646266345835813.
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博士<br>國立中央大學<br>資訊管理研究所<br>96<br>In the last decade, the problem of getting a consensus group ranking from users’ ranking data has received increased attention due to its widespread applications. Previous research solved this problem by consolidating the opinions of all users, thereby obtaining an ordering list of all items that represent the achieved consensus. The weakness of this approach, however, is that it always produces a ranking list of all items, regardless of how many conflicts exist among users. This work rejects the forced agreement of all items. Instead, we define a new concept, maximum consensus sequences, which are the longest ranking lists of items that agree with the majority and disagree only with the minority. Based on this concept, we use two kinds of input data, individual’s total ranking and individual’s partial rankings, to develop algorithms to discover maximum consensus sequences and also to identify conflict items that need further negotiation. Besides, we propose another algorithm to achieve personalized rankling list which can be used in recommender system. Extensive experiments are carried out using synthetic data sets, and the results indicate that the proposed methods are computationally efficient.
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Chang, Jerry, and 張立群. "Consensus Cis-sequences of Purified Trans-factors from Yeast Expression System." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/97186947453766587193.
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碩士<br>中國文化大學<br>生物科技研究所<br>93<br>The goal of this thesis is to establish fast and global analysis platform of differential regulomics , it continue futher study with probe of labeling human tumor cell line to screening human liver complementary nucleic acid from high expressing and specific yeast yield to obtain Trans -factors, and then utilize existing Trans -factors selection regulation relevantly to the Cis element of antitumor gene p53 by building and constructing the screening regulomics technological integrality entirely. This research is purification transcription factor p53 protein from transform yeast cell line with Cyclic Amplification and Selection of Targets to obtain nucleic acid, finding consensus sequence of transcription factor binding from one hybride selection system,verify that adjusts regulomics subduction type and distinguishes the feasibility which analyses the technological platform and improves the relatively quick-acting and specific.
In the building of protein purification system , utilize HQ column of strangly basic anion exchanger, -N+(CH3)3 to bind protein and then utilize different concentration of salt solvent to elute, and receive the goal protein, namely is purification transcription factor protein p53 by the expression yeast,by this protein CASTing the nucleic acids, selection complex of CASTing nucleic acid and transcription factor, obtain specific transcription factor of CASTing nucleic acid template , insert nucleic acid template to construct on b-gal reporter gene pLacZi of yeast , transform to the expressing transcription factor yeast competent cell , plating and X-gal blue- white selection, the transformation success yeast could make gene expression yield β-galactosidase to destroy X-gal and blue response, sequenceing CASTing nucleic acid from blue response yeast of clone construct reporter plasmid , compare and analysing to obtaining the consensus sequence.
The thesis study has accomplished the preliminart feasibility of the differential subtractive analysis of global regulomics with performance features on simultaneous identification of multiple unknown targets at higher speed .
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Wang, Pei-Hsueh, and 王碧雪. "Yeast One-Hybrid Screening System forFinding Consensus Cis-sequences of Expressed Trans-factors." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/39649472719114590968.
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碩士<br>中國文化大學<br>生物科技研究所<br>93<br>Summary
The goal in research of this thesis, in order to use known p53 protein to prove that innovate the technological platform, combine the saccharomycete form and mix the system of shutting (Yeast One-Hybrid System) increase and choose with the circulation type goal (Cyclic Amplification and Selection of Targets, the mutual experiment application that CASTing ), fetch its safe effective advantage and obtain specific p53 protein uniqueness and combine the gene order, use the transcription factor activating area of p53 to form the characteristic with the single-minded key of combining area of DNA, get and combine the array (Consensus binding seequence ) with the common understanding to the regulation and control gene order of the side transcription factor p53 protein, it is unable to measure the shortcomings of a lot of unknown goals and experiments consuming time at the same time to improve the traditional method, it is higher than the short , uniqueness while offering taking, the technological platform of the innovation that a lot of goals measure at the same time
This research is by displaying the bacterial strain to side transcription factor (Trans ) and saccharomycete, recombinate the circulation type goal to increase and choose (Cyclic Amplification and Selection of Targets, CASTing), nucleic acid template report gene carrier system, is it is it is it is it shut system is it is it collect CASTing nucleic acid to check the storehouse combine arrays to prove to screen to mix only to use to innovate to set up, obtained the array by the parallelism of the array and should combine with side to the common understanding of the side protein. Use and mix and equal to the system and screen p53 and display a relevant CASTing nucleic acid to side transcription factor and saccharomycete only, prove conclusively CASTing nucleic acid template person who distinguish and with side array person who adjust and control, utilize p53-CASTing nucleic acid and pLacZi quality body is it is it shut systematic | Â to mix only to set up - gal report to is it check the storehouse to collect with side array carrier, transfer to shape p53 and display to side transcription factor and saccharomycete the bacterial strain, carry on X-gal and screen the system bluly and whitly and confirm being selected the bone CSATing nucleic acid array. Choosing the blue colony displayed to the side p53 transcription factor, the preface got and adjusted and controlled arrays to the side transcription factor definitely, carrying on arrays will combine the common understanding array than to obtaining p53 protein , reach and set up and verify this technological platform.
Key word: Adjust and control the gene order, common understanding combine the array , p53 protein , circulation type goal and increase and choose , the saccharomycete form mixes and shuts the system , screens the system bluly and whitly
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Hsu, Chien-Hsiu, and 徐千琇. "Identification of the consensue sequence of LexA binding site in Xanthomonas campestris pv. citri." Thesis, 2002. http://ndltd.ncl.edu.tw/handle/61257460262365186230.
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碩士<br>輔仁大學<br>生物學系<br>90<br>Abstract
A Xanthomonas campestris gene cluster consisted of lexA, recA and recX genes was identified and characterized. Electrophoretic mobility shift analysis indicated that the LexA protein of X. c. pv. citri bind to the lexA and recA promoters of X. c. pv. campestris and X. oryzae. pv. oryzae and repress the Xanthomonas campestris recA gene, indicating that the X. c. pv. citri LexA protein was functional in different Xanthomonas campestris pathovars. It was confirmed that a symmetrical sequence of TTAGTAGTAATACTACTAA within the lexA promoter region is essential for the LexA protein binding by site-directed mutagenesis and electrophoretic mobility shift analysis. A lexA mutated promoter increased in the transcriptional and translational level, due to loss of the binding ability of the LexA protein. However, the LexA protein was able to bind to the similar sequences of the recA and recX promoters. It was suggested that the consensus sequence of LexA binding in X. c. pv. citri is TTAGTAGTAATACTACTAA.
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Wentzel, Johannes Frederik. "Investigating the importance of co-expressed rotavirus proteins in the development of a selection-free rotavirus reverse genetics system / Johannes Frederik Wentzel." Thesis, 2014. http://hdl.handle.net/10394/12270.
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Reverse genetics is an innovative molecular biology tool that enables the manipulation of
viral genomes at the cDNA level in order to generate particular mutants or artificial viruses.
The reverse genetics system for the influenza virus is arguably one of the best illustrations of
the potential power of this technology. This reverse genetics system is the basis for the
ability to regularly adapt influenza vaccines strains. Today, reverse genetic systems have
been developed for many animal RNA viruses. Selection-free reverse genetics systems have
been developed for the members of the Reoviridae family including, African horsesickness
virus, bluetongue virus and orthoreovirus. This ground-breaking technology has led to the
generation of valuable evidence regarding the replication and pathogenesis of these viruses.
Unfortunately, extrapolating either the plasmid-based or transcript-based reverse genetics
systems to rotavirus has not yet been successful. The development of a selection-free
rotavirus reverse genetics system will enable the systematic investigation of poorly
understood aspects of the rotavirus replication cycle and aid the development of more
effective vaccines, amongst other research avenues.
This study investigated the importance of co-expressed rotavirus proteins in the
development of a selection-free rotavirus reverse genetics system. The consensus
sequences of the rotavirus strains Wa (RVA/Human-tc/USA/WaCS/1974/G1P[8]) and SA11
(RVA/Simian-tc/ZAF/SA11/1958/G3P[2]) where used to design rotavirus expression
plasmids. The consensus nucleotide sequence of a human rotavirus Wa strain was
determined by sequence-independent cDNA synthesis and amplification combined with
next-generation 454® pyrosequencing. A total of 4 novel nucleotide changes, which also
resulted in amino acid changes, were detected in genome segment 7 (NSP3), genome
segment 9 (VP7) and genome segment 10 (NSP4). In silico analysis indicated that none of
the detected nucleotide changes, and consequent amino acid variations, had any significant
effect on viral structure. Evolutionary analysis indicated that the sequenced rotavirus WaCS
was closely related to the ParWa and VirWa variants, which were derived from the original
1974 Wa isolate. Despite serial passaging in animals, as well as cell cultures, the Wa genome
seems to be stable. Considering that the current reference sequence for the Wa strain is a
composite sequence of various Wa variants, the rotavirus WaCS may be a more appropriate
reference sequence.
The rotavirus Wa and SA11 strains were selected for plasmid-based expression of rotavirus
proteins, under control of a T7 promoter sequence, due to the fact that they propagate well
in MA104 cells and the availability of their consensus sequences. The T7 RNA polymerase
was provided by a recombinant fowlpox virus. After extensive transfection optimisation on a
variety of mammalian cell lines, MA104 cells proved to be the best suited for the expression
rotavirus proteins from plasmids. The expression of rotavirus Wa and SA11 VP1, VP6, NSP2
and NSP5 could be confirmed with immunostaining in MA104 and HEK 293H cells. Another
approach involved the codon-optimised expression of the rotavirus replication complex
scaffold in MA104 cells under the control of a CMV promoter sequence. This system was
independent from the recombinant fowlpox virus. All three plasmid expression sets were
designed to be used in combination with the transcript-based reverse genetics system in
order to improve the odds of developing a successful rotavirus reverse genetics system. Rotavirus transcripts were generated using transcriptively active rotavirus SA11 double
layered particles (DLPs). MA104 and HEK293H cells proved to be the best suited for the
expression of rotavirus transcripts although expression of rotavirus VP6 could be
demonstrated in all cell cultures examined (MA104, HEK 293H, BSR and COS-7) using
immunostaining. In addition, the expression of transcript derived rotavirus VP1, NSP2 and
NSP5 could be confirmed with immunofluorescence in MA104 and HEK 293H cells. This is
the first report of rotavirus transcripts being translated in cultured cells. A peculiar cell
death pattern was observed within 24 hours in response to transfection of rotavirus
transcripts. This observed cell death, however does not seem to be related to normal viral
cytopathic effect as no viable rotavirus could be recovered. In an effort to combine the
transcript- and plasmid systems, a dual transfection strategy was followed where plasmids
encoding rotavirus proteins were transfected first followed, 12 hours later, by the
transfection of rotavirus SA11 transcripts. The codon- optimised plasmid system was
designed as it was postulated that expression of the DLP-complex (VP1, VP2, VP3 and VP6),
the rotavirus replication complex would form and assist with replication and/or packaging.
Transfecting codon- optimized plasmids first noticeably delayed the mass cell death
observed when transfecting rotavirus transcripts on their own. None of the examined coexpression
systems were able to produce a viable rotavirus.
Finally, the innate immune responses elicited by rotavirus transcripts and plasmid-derived
rotavirus Wa and SA11 proteins were investigated. Quantitative RT-PCR (qRT-PCR)
experiments indicated that rotavirus transcripts induced high levels of the expression of the
cytokines IFN- α1, IFN-1β, IFN-λ1 and CXCL10. The expression of certain viral proteins from
plasmids (VP3, VP7 and NSP5/6) was more likely to stimulate specific interferon responses,
while other viral proteins (VP1, VP2, VP4 and NSP1) seem to be able to actively suppress the
expression of certain cytokines. In the light of these suppression results, specific rotavirus
proteins were expressed from transfected plasmids to investigate their potential in
supressing the interferon responses provoked by rotavirus transcripts. qRT-PCR results
indicated that cells transfected with the plasmids encoding NSP1, NSP2 or a combination of
NSP2 and NSP5 significantly reduced the expression of specific cytokines induced by
rotavirus transcripts. These findings point to other possible viral innate suppression
mechanisms in addition to the degradation of interferon regulatory factors by NSP1. The
suppression of the strong innate immune response elicited by rotavirus transcripts might
well prove to be vital in the quest to better understand the replication cycle of this virus and
eventually lead to the development of a selection-free reverse genetics system for rotavirus.<br>PhD (Biochemistry), North-West University, Potchefstroom Campus, 2014