Showing papers on "Upstream activating sequence published in 2014"

The Mediator complex: a master coordinator of transcription and cell lineage development

Abstract: Mediator is a multiprotein complex that is required for gene transcription by RNA polymerase II. Multiple subunits of the complex show specificity in relaying information from signals and transcription factors to the RNA polymerase II machinery, thus enabling control of the expression of specific genes. Recent studies have also provided novel mechanistic insights into the roles of Mediator in epigenetic regulation, transcriptional elongation, termination, mRNA processing, noncoding RNA activation and super enhancer formation. Based on these specific roles in gene regulation, Mediator has emerged as a master coordinator of development and cell lineage determination. Here, we describe the most recent advances in understanding the mechanisms of Mediator function, with an emphasis on its role during development and disease.

Kin28 regulates the transient association of Mediator with core promoters

Abstract: Mediator is an essential, broadly utilized eukaryotic transcriptional co-activator. How and what it communicates from activators to RNA polymerase II (RNAPII) remains an open question. Here we performed genome-wide location profiling of Saccharomyces cerevisiae Mediator subunits. Mediator is not found at core promoters but rather occupies the upstream activating sequence (UAS), upstream of the pre-initiation complex. In the absence of Kin28 (CDK7) kinase activity, or in cells where the RNAPII C-terminal domain (CTD) is mutated to replace Ser5 with alanines, however, Mediator accumulates at core promoters together with RNAPII. We propose that Mediator is quickly released from promoters upon Ser5 phosphorylation by Kin28 (CDK7), which also allows for RNAPII to escape from the promoter.

Differential acetylation of histone H3 at the regulatory region of OsDREB1b promoter facilitates chromatin remodelling and transcription activation during cold stress.

Abstract: The rice ortholog of DREB1, OsDREB1b, is transcriptionally induced by cold stress and over-expression of OsDREB1b results in increase tolerance towards high salt and freezing stress. This spatio-temporal expression of OsDREB1b is preceded by the change in chromatin structure at the promoter and the upstream region for gene activation. The promoter and the upstream region of OsDREB1b genes appear to be arranged into a nucleosome array. Nucleosome mapping of ∼700bp upstream region of OsDREB1b shows two positioned nucleosomes between −610 to −258 and a weakly positioned nucleosome at the core promoter and the TSS. Upon cold stress, there is a significant change in the nucleosome arrangement at the upstream region with increase in DNaseI hypersensitivity or MNase digestion in the vicinity of cis elements and TATA box at the core promoter. ChIP assays shows hyper-acetylation of histone H3K9 throughout the locus whereas region specific increase was observed in H3K14ac and H3K27ac. Moreover, there is an enrichment of RNA PolII occupancy at the promoter region during transcription activation. There is no significant change in the H3 occupancy in OsDREB1b locus negating the possibility of nucleosome loss during cold stress. Interestingly, cold induced enhanced transcript level of OsDREB1b as well as histone H3 acetylation at the upstream region was found to diminish when stressed plants were returned to normal temperature. The result indicates absolute necessity of changes in chromatin conformation for the transcription up-regulation of OsDREB1b gene in response to cold stress. The combined results show the existence of closed chromatin conformation at the upstream and promoter region of OsDREB1b in the transcription “off” state. During cold stress, changes in region specific histone modification marks promote the alteration of chromatin structure to facilitate the binding of transcription machinery for proper gene expression.

A targeted gene expression system using the tryptophan repressor in zebrafish shows no silencing in subsequent generations

Abstract: The ability to visualize and manipulate cell fate and gene expression in specific cell populations has made gene expression systems valuable tools in developmental biology studies. Here, we describe a new system that uses the E. coli tryptophan repressor and its upstream activation sequence (TrpR/tUAS) to drive gene expression in stable zebrafish transgenic lines and in mammalian cells. We show that TrpR/tUAS transgenes are not silenced in subsequent generations of zebrafish, which is a major improvement over some of the existing systems, such as Gal4/gUAS and the Q-system. TrpR transcriptional activity can be tuned by mutations in its DNA-binding domain, or silenced by Gal80 when fused to the Gal4 activation domain. In cases in which more than one cell population needs to be manipulated, TrpR/tUAS can be used in combination with other, existing systems.

A novel approach to propagate flavivirus infectious cDNA clones in bacteria by introducing tandem repeat sequences upstream of virus genome.

Abstract: Despite tremendous efforts to improve the methodology for constructing flavivirus infectious cDNAs, the manipulation of flavivirus cDNAs remains a difficult task in bacteria. Here, we successfully propagated DNA-launched type 2 dengue virus (DENV2) and Japanese encephalitis virus (JEV) infectious cDNAs by introducing seven repeats of the tetracycline-response element (7×TRE) and a minimal cytomegalovirus (CMVmin) promoter upstream of the viral genome. Insertion of the 7×TRE-CMVmin sequence upstream of the DENV2 or JEV genome decreased the cryptic E. coli promoter (ECP) activity of the viral genome in bacteria, as measured using fusion constructs containing DENV2 or JEV segments and the reporter gene Renilla luciferase in an empty vector. The growth kinetics of recombinant viruses derived from DNA-launched DENV2 and JEV infectious cDNAs were similar to those of parental viruses. Similarly, RNA-launched DENV2 infectious cDNAs were generated by inserting 7×TRE-CMVmin, five repeats of the GAL4 upstream activating sequence, or five repeats of BamHI linkers upstream of the DENV2 genome. All three tandem repeat sequences decreased the ECP activity of the DENV2 genome in bacteria. Notably, 7×TRE-CMVmin stabilized RNA-launched JEV infectious cDNAs and reduced the ECP activity of the JEV genome in bacteria. The growth kinetics of recombinant viruses derived from RNA-launched DENV2 and JEV infectious cDNAs displayed patterns similar to those of the parental viruses. These results support a novel methodology for constructing flavivirus infectious cDNAs, which will facilitate research in virology, viral pathogenesis and vaccine development of flaviviruses and other RNA viruses.

Monitoring autophagy in Drosophila using fluorescent reporters in the UAS-GAL4 system.

Abstract: Following autophagy induction, the autophagy-related protein Atg8 undergoes ubiquitin-like conjugation to phosphatidylethanolamine and inserts into the autophagosome membrane. Transgenic Drosophila lines expressing Drosophila Atg8 (DrAtg8a) fused to green fluorescent protein (GFP), mCherry, or dual-tagged GFP-mCherry have been constructed and are extremely useful for monitoring autophagy. The use of GFP-mCherry-Atg8a is particularly advantageous because it allows for the assessment of autophagy induction as well as autophagy flux. GFP-mCherry-Atg8a fluoresces yellow in nonacidic structures including the autophagosome. When autophagosomes fuse with lysosomes to form autolysosomes, GFP fluorescence is quenched by the acidic hydrolases and the resulting autolysosome will fluoresce red. The upstream activating sequence (UAS)-GAL4 system allows for the ectopic expression of the gene of interest (in this case, DrAtg8a) in a tissue-specific manner. Here we provide a protocol for monitoring autophagic flux by fluorescence microscopy by expressing UASp-GFP-mCherry-DrAtg8a in the Drosophila germline.

Spacing requirements for Class I transcription activation in bacteria are set by promoter elements

Abstract: The Escherichia coli cAMP receptor protein (CRP) activates transcription initiation at many promoters by binding upstream of core promoter elements and interacting with the C-terminal domain of the RNA polymerase α subunit. Previous studies have shown stringent spacing is required for transcription activation by CRP. Here we report that this stringency can be altered by the nature of different promoter elements at target promoters. Several series of CRP-dependent promoters were constructed with CRP moved to different upstream locations, and their activities were measured. The results show that (i) a full UP element, located immediately downstream of the DNA site for CRP, relaxes the spacing requirements for activation and increases the recruitment of RNAP and open complex formation; (ii) the distal UP subsite plays the key role in this relaxation; (iii) modification of the extended −10 element also affects the spacing requirements for CRP-dependent activation. From these results, we conclude that the spacing requirements for CRP-dependent transcription activation vary according to the sequence of different promoter elements, and our results are important for understanding the organization of promoters in many different bacteria which are controlled by transcription factors that use activatory mechanisms similar to CRP.

Cloning and Characterization of the Human Trefoil Factor 3 Gene Promoter

Abstract: Human trefoil factor 3 (hTFF3) is a small-molecule peptide with potential medicinal value. Its main pharmacological function is to alleviate gastrointestinal mucosal injuries caused by various factors and promote the repair of damaged mucosa. However, how its transcription is regulated is not yet known. The aim of this study was to clone the hTFF3 gene promoter region, identify the core promoter and any transcription factors that bind to the promoter, and begin to clarify the regulation of its expression. The 5′ flanking sequence of the hTFF3 gene was cloned from human whole blood genomic DNA by PCR. Truncated promoter fragments with different were cloned and inserted into the pGL3-Basic vector to determine the position of the core hTFF3 promoter. Transcription element maintaining basic transcriptional activity was assessed by mutation techniques. Protein-DNA interactions were analyzed by chromatin immunoprecipitation (ChIP). RNA interference and gene over-expression were performed to assay the effect of transcription factor on the hTFF3 expression. The results showed that approximately 1,826 bp of the fragment upstream of hTFF3 was successfully amplified, and its core promoter region was determined to be from −300 bp to −280 bp through analysis of truncated mutants. Mutation analysis confirmed that the sequence required to maintain basic transcriptional activity was accurately positioned from −300 bp to −296 bp. Bioinformatic analysis indicated that this area contained a Sp1 binding site. Sp1 binding to the hTFF3 promoter was confirmed by ChIP experiments. Sp1 over-expression and interference experiments showed that Sp1 enhanced the transcriptional activity of the hTFF3 promoter and increased hTFF3 expression. This study demonstrated that Sp1 plays an important role in maintaining the transcription of hTFF3.

Lights and Larvae: Using Optogenetics to Teach Recombinant DNA and Neurobiology

Abstract: [ILLUSTRATION OMITTED] Switching genes between organisms and controlling an animal's brain using lasers may seem like science fiction, but with advancements in a technique called optogenetics, such experiments are now common in neuroscience research. Optogenetics combines recombinant DNA technology with a controlled light source to help researchers address biomedical questions in the life sciences. The technique has gained the most traction in neurobiology--the biology of the nervous system--where specific wavelengths of light are used to control or measure the activity of neurons in transgenic organisms (i.e., those with artificially inserted genes). These optical recording and stimulation techniques are used in nervous system preparations ranging from individual cells in culture to whole organisms, where the observations and data collected have been used to determine which neurons are involved in specific animal behaviors. In this article, we describe an inexpensive Drosophila (fruit fly) optogenetics experiment used to teach principles of the nervous system, genetics, and bioengineering at the high school level. (See sidebar [p. 44] for specific learning objectives and key concepts for the lab.) Fruit flies receive an algae gene Algae and other microorganisms have been known to sense and emit light (Foster and Smyth 1980). Advances in molecular biology techniques near the end of the 20th century enabled researchers to determine which proteins were involved in phototaxis (movement in response to light), clone the respective genes, and transfer them into new species for research. The proteins themselves are called channelrhodopsins, which are transmembrane ion channels that convey a non-specific ion flux when the channel is activated by a specific wavelength of light. Channelrhodopsin-2 (ChR2) is sensitive to blue light. By introducing point mutations into the gene, researchers have altered the light sensitivity and optimized ion conduction in these channels, making photo-activation more efficient and easier to use. These advances paved the way for optogenetics to be implemented in teaching laboratories. The basic methods and concepts for using optogenetics in the undergraduate classroom have been developed by Pulver and colleagues (Pulver et al. 2011a; Pulver et al. 2011b; Pulver and Berni 2012). Here we have adapted those exercises to a high school--level module that addresses core disciplinary ideas in the Next Generation Science Standards (NGSS Lead States 2013) (Figure 1). Expression of ChR2 in Drosophila melanogaster is restricted to motor neurons A huge breakthrough in Drosophila transgenics occurred when the yeast GAL4-UAS binary expression system was introduced into the fly genome (Brand and Perrimon 1993; Duffy 2002). Thus the transgenic ChR2 could be expressed in specific subsets of cells using this standard binary expression system. The ChR2 transgene is controlled by the yeast upstream activation sequence (UAS) for a galactose-induced transcription factor (GAL4), meaning that the transgene is carried in every cell but is only expressed where GAL4 is expressed. Expression of the GAL4 gene is controlled by a promoter sequence either from a nearby promoter in the fly's genome or a specific promoter that is added with the GAL4 transgene sequence. By adding a promoter to the GAL4 sequence that is expressed specifically in the nervous system (e.g., a gene that codes for an enzyme involved in synthesis of a neurotransmitter), the GAL4 gene only gets expressed in the nervous system, thus activating expression of the ChR2 transgene specifically in the nervous system. The promoter used to drive expression of GAL4 in the flies used for this activity is called OK371-GAL4. The promoter element is from the Drosophila vesicular glutamate transporter gene (DVGLUT) that is expressed almost exclusively in neurons that release glutamate (Mahr and Aberle 2006). …

Interaction between Transactivation Domain of p53 and Middle Part of TBP-Like Protein (TLP) Is Involved in TLP-Stimulated and p53-Activated Transcription from the p21 Upstream Promoter

Abstract: TBP-like protein (TLP) is involved in transcriptional activation of an upstream promoter of the human p21 gene. TLP binds to p53 and facilitates p53-activated transcription from the upstream promoter. In this study, we clarified that in vitro affinity between TLP and p53 is about one-third of that between TBP and p53. Extensive mutation analyses revealed that the TLP-stimulated function resides in transcription activating domain 1 (TAD1) in the N-terminus of p53. Among the mutants, #22.23, which has two amino acid substitutions in TAD1, exhibited a typical mutant phenotype. Moreover, #22.23 exhibited the strongest mutant phenotype for TLP-binding ability. It is thus thought that TLP-stimulated and p53-dependent transcriptional activation is involved in TAD1 binding of TLP. #22.23 had a decreased transcriptional activation function, especially for the upstream promoter of the endogenous p21 gene, compared with wild-type p53. This mutant did not facilitate p53-dependent growth repression and etoposide-mediated cell-death as wild-type p53 does. Moreover, mutation analysis revealed that middle part of TLP, which is requited for p53 binding, is involved in TLP-stimulated and p53-dependent promoter activation and cell growth repression. These results suggest that activation of the p21 upstream promoter is mediated by interaction between specific regions of TLP and p53.

The structural properties of DNA regulate gene expression

Abstract: Regulatory sequences such as promoters not only contain cis-regulatory elements as switches of transcription, but also exhibit particular topological features. In this paper, we introduce a systematic genome scale approach to characterize the roles of structural conformation and stability profile of promoter sequence in gene expression. The average free energy of promoter dinucleotides stacking nearest neighbors are subjected to scrutiny by statistical hidden Markov models to reveal the function of constrains and properties of promoter structure in transcription. When applied for a 1000 bp 5′ upstream sequence of genes, the proposed model via assessing free energy profile identified co-expressed genes of Arabidopsis thaliana in response to the auxin hormone. The applied perspective dynamic network which mediates transcription regulation provides a great hindrance to conceive how DNA conformation interacts with cis-regulatory elements, chromatin structure and many other factors. This study indeed drew the complexity of the promoter's regulatory behavior from sequence over the former studies and evokes a new hypothesis to be validated experimentally.

Bioinformatic analysis of the hsa-miR-1908 upstream promoter region

Abstract: Objective To predict the functions of hsa-miR-1908 promoter using various bioinformatic tools,and to provide clues for further study on transcriptional regulation mechanism of miR-1908 in human adipocytes.Methods The promoter sequence of miR-1908 was obtained from Ensemble,and then the CpG islands and transcription factor binding sites were predicted by a variety of online bioinformatic tools.Results The length of the miR-1908 promoter sequence was 1 458 bp.The CpG islands,which inhibited the transcription of miR-1908,were located at(438-756) bp,(836-937) bp and(979-1374) bp.Meanwhile,15 transcription factor binding sites were found in the promoter sequence of miR-1908.Conclusions miRNA upstream promoter related bioinformatics can not only improve the efficiency of microRNA promoter research,but also provide further important information on transcriptional regulation of miR-1908.

Evolution of transcriptional regulation in "Escherichia coli"

Abstract: During gene expression, transcription initiation marks the first step towards synthesis of functional proteins. Expression levels of specific types of RNA molecules in the cell depend on the underlying genotype of the promoter sequence. Prediction of expression levels from the promoter sequence alone can have important implications for the design of artificial promoters. In this work, we explored promoter determinants that cause differences in expression levels and tracked how a certain level can be reached by a directed evolution experiment in E.coli. Promoter sequences were evolved from a million random sequences with selection on expression level and high mutation rate. Mapping of expression phenotypes to the underlying promoter genotypes revealed what sequence features determine the rate of transcription. If no differential expression is required, incorporation of Sigma70 binding sites allows expression. However, predicted affinity of Sigma70 to bind to a promoter sequence in different promoter contexts is not explanatory in terms of expression levels, suggesting that other sequence features determine the rate of transcription. Furthermore, separation of functional promoter sequences to non-regulatory sequences is promoted by high AT content as well as preference of generally longer promoter sequences. Recovery of an essential missing gene function can also be obtained by overexpression of other genes present in the genome by changing the strength of Sigma70 binding to the promoter sequence. Small changes in the expression level were shown to have a severe impact on the fitness of the organism. The amount of deviation away from the optimal expression level in clonal promoter populations has been shown to depend on the promoter’s genotype. We are presenting an evolutionary model to explain under which regulatory settings selection favors high variance in expression levels between cells.

Method for quickly constructing high-expression and stable cell strains

Abstract: The invention provides a method for quickly constructing high-expression and stable cell strains. The method comprises the following steps: (1) constructing an expression vector plasmid of Ga14-VP16 fusion proteins; (2) constructing an expression vector plasmid of a UAS (upstream activating sequence)-target gene proteins; (3) transducing the expression vector plasmid of Ga14-VP16 fusion proteins and the expression vector plasmid of the UAS-target gene proteins into host cells; (4) selecting and screening stably expressed cell strains of target genes; (5) performing multiplication; (6) obtaining the high-expression and stable cell strains. The method disclosed by the invention can be used for effectively constructing the high-expression and stable cell strains.

New tool for late stage visualization of Oskar protein in Drosophila melanogaster oocytes

Abstract: Determination of the anterior/posterior axis in Drosophila melanogaster is established in oogenesis, in part by localization of the oskar (osk) mRNA to the posterior pole of the oocyte. This mRNA is subject to translational control to ensure that it is translated only when properly localized, but the process is not fully elucidated. In identifying genes with roles in translational control of osk, antibody staining is typically used to assay Osk expression. A limitation of this method is that the vitelline membrane, which surrounds the oocyte and is deposited during oogenesis, prevents access to antibodies, and so later stages of Osk expression cannot be monitored. Although early work focused on the the stage when Osk protein first appears, we now know that the majority of Osk is translated in later stages of oogenesis. My goal was to look for new factors involved in osk translational control by monitoring Osk::GFP expression and ‘knocking down’ the expression of candidate genes by using RNA interference (RNAi). An expansive collection of transgenic RNAi (TRiP) lines target a large fraction of all Drosophila genes. Knockdown is achieved by crossing a chosen TRiP line to a GAL4 driver (MAT3), which directs TRiP expression in the germline cells of the ovary. Knockdowns can be more useful than mutants, which are often homozygous lethal or arrest oogenesis earlier than the stage we wish to visualize. Knockdowns typically partially decrease the expression of a target gene, thus avoiding the complications associated with a null mutant. To simplify the screen I recombined the MAT3 driver and osk::GFP transgene on to the same chromosome. This allowed me to test each TRiP line with a single cross, with the TRiP transgene from one parent and osk::GFP and MAT3 from the other parent. To make the recombinant I crossed MAT3 and osk::GFP flies, and used a PCR assay to test the progeny of individual candidates for presence of MAT3 and Osk::GFP. To validate the knockdown approach, I crossed the osk::GFP MAT3 recombinant TRiP lines of known regulators of osk mRNA to see if I obtained the same phenotypes as previously observed for mutants of these genes. It was found that these knockdowns show no change in expression of Oskar::GFP protein in TRiP lines of activators and increased expression in TRiP lines of repressors, except for that of hephaestus, which exhibited decreased expression. vasa, which is required for a late stage in Osk expression, also appears to have decreased expression. In the future, we plan to use this recombinant in crosses with other TRiP lines of known genes used in osk translational control such as tudor, orb, and bruno. The knockdown of hephaestus and aubergine should be reperformed in order to ensure that the results are accurate. In addition, this recombinant can be used in screens to find genes involved in osk expression during late stage oogenesis so that more detailed mechanisms of control in posterior development can be described. Background RNA interference RNA interference is a natural biological process which has been repurposed as a tool in functional genomics. This gene silencing mechanism hinges on the transcription of hairpin RNAs (hpRNAs) which fold upon themselves to form double stranded DNA (dsDNA). These dsDNA strands are then processed by the RNA-induced silencing complex (RISC), which cleaves the dsDNA into siRNAs. These siRNAs are highly specific to an mRNA sequence and upon binding to the target mRNA, will direct the sequence-specific degradations of this mRNA strand (Kuttenkeuler and Boutros, 2004). Within functional genomics, an Upstream Activation Sequence (UAS) has been added to the promoter of the hpRNA sequence to ensure that its transcription is dependent on the presence of a GAL4 driver, which specifically binds to the UAS to activate gene transcription. This system allows for tissue or cell specific activation of the RNAi, by constructing a GAL4 driver with a promoter that is activated in that specific tissue or cell. Once the RNAi gene and the GAL4 driver are located in the same genome, the activated RNAi will generate a knockdown to partially or completely diminish the target gene expression in the desired tissue. In doing this, lethal effects of diminished expression in other tissues or cells can be avoided. Stable stocks of flies that use this transgenic RNAi are known as TRiP lines and can target a large number of Drosophila genes. Collection of TRiP Lines Utilized In order to ensure that the osk::GFP MAT3 recombinant didn’t have any unexpected interactions with many of the proteins and mRNAs involved in translational control of osk, TRiP lines of major known regulators of osk mRNA were selected for screening. These included repressors such as Bicaudal-C (Bic-C)hephaestus (heph), and Argonaute-1 (AGO1) and activators such as vasa and staufen. RNA binding proteins such as Staufen (Stau) and Hephaestus (Heph) are found in the same RNP particles that osk mRNA travels in and mediate translational control of osk mRNA (Martin et al. 2003). Staufen proteins mediate posterior localization of osk, as stau mutants develop osk particles in the anterior margin of the oocyte (Irion et al. 2006). Heph, also known as a polypyrimidine tract binding (PTB) protein, has shown the ability to bind to osk mRNA in a sequence specific manner and is necessary for the translational repression of this mRNA while it is in the process of localization (Besse et al. 2009). The repressor Bic-C is known to be a part of translational control and the absence of BicC in oocytes yields premature osk translation prior to its posterior localization (Saffman et al. 1998). Another repressor, argonaute, is thought to be involved in the osk repression but its role is uncertain. The activator aubergine (aub) is necessary for posterior development and osk activation once it reaches the posterior pole (Harris and Macdonald, 2001). Figure 1. Wild type expression of endogenous osk mRNA in a D. melanogaster oocyte traveling from the nurse cells and into the oocyte before being localized at the posterior pole. (Gonsalvez and Long, 2012) Each of these is necessary for appropriate expression of the osk mRNA in its repression, localization, and activation at the posterior pole of the oocyte. One can see the orientation of this mRNA within a developing oocyte (Stage 10A) in Figure 1 Results The recombinant line of flies carrying both the GFP-tagged oskar and a GAL-4 driver required the crossing of flies with the transgene for GFP-tagged oskar (9173) and those carrying the GAL4 driver MAT3. Using the cross w; 9173 TM3Sb ×w; TM2 flies of the genotype w; 9173 MAT3 were generated. Females of this genotype were collected to cross with wild type (w118) males. The resulting flies were screened to find recombinant males with the predicted eye color specific for w; ! , which were then crossed to females of doubly balanced lines to generate a stable line of flies with both the transgene for GFP-tagged Oskar and the GAL4 driver. The recombinant males were then removed from the population and tested by mixing the fly sample DNA with target oligonucleotides and performing PCR to amplify the target DNA if the gene is in the sample. The results are shown in Figure 2 and indicate that four of the five potential recombinants contained both MAT3 and 9173 genes. Of these, the third recombinant had a more robust population and was therefore chosen as the stock population for the remaining tests. Figure 2. Agarose gel displaying the presence or absence of the desired genes using oligonucleotides specific for the GFP-tagged oskar and the GAL4 driver MAT3. Potential recombinants are described as R1, R2, R3, R4, and R5. The recombinant was crossed to the TRiP lines of hephaestus, vasa, staufen, aubergine, Argonaute-1 and Bicaudal-C to drive RNAi activity and generate knockdowns of these target genes and observe any changes in Oskar expression. The results can be seen in Figure 3.