From Text A, The Father In The Family Has Two Opposite Personality. He

From text A, the father in the family has two opposite personality. He is a cheerful person in his workplace however when come to home, he misbehaves. In line 21, ?he was like the scotch in the smooth, happy machine of the home?. In line 51, ?They united with in the work? His son felled that his father does even bothers about him and the family. However in line 25, stated that he would dearly have liked the children to talk to him, but they could not. From text C, the problem occurs from the son, Albert. He is a kind like to show a rebellion to his mother. He feels that he own his freedom. He can do whatever wants. Just because he is the only person his mother life, she tend to chow how much she care about him. In my opinion, Albert may be overshadowed by his late father whom I feel behave strictly and controlling him too much. Since his father has passed away, he believes that now it is the payback time.

How successful was Peels Ministry 1841-1846 Essays

How successful was Peels Ministry 1841-1846 Essays How successful was Peels Ministry 1841-1846 Essay How successful was Peels Ministry 1841-1846 Essay Peels ministry essentially benefited the nation as a whole, which would suggest that Peels ministry was very successful. However, in doing so he split the party we had worked so hard to bring together. Therefore when considering whether his ministry was a success or not it has to be assessed whether a ministry has to primarily look after the partys interests or the nations. Most historians including the likes of Donald Read take the view that the nations interests should be the priority, and this is why Peels ministry is considered successful. Despite diluting some of the Traditional Tory views which agitated the landowners, and eventually splitting the party through the Repeal of the Corn Laws in 1846 Peels ministry improved the living and working conditions of the average person. He managed to do this most importantly through his financial reforms which were very successful, as well as his social reforms. Peel aimed to be fairer to the poor, although at some expense of the landowners. The decline of radical groups within Britain though out Peels ministry marked its success, and although he did carry out a number of unsuccessful polices in Ireland, he critically prevented them from leaving the Union through good work in 1843. Peels financial reforms were the most important reasons for its success despite their unpopularity in some sections of the party. In 1941 he inherited  £7million of debt from the Whig government before him, but through a selection of significant reforms, most notably the 1842 Budget he was able to boast a  £5 million surplus by the end of 1865. Much of the financial reform that Peels ministry passed could be considered as social reform too, which was very important in improving the conditions for people in Britain. The 1842 Budget aimed to appeal to the rich through a sense of justice and self preservation, at the same time as showing the poor there was a fairer system. Peel boldly reintroduced income tax for 3years which affected those wi th annual incomes of  £150 or more. This was hugely unpopular with the landowners, and therefore a great section of his parties support base however, it was greatly successful and despite being expected to raise 3-4 million a year it actually raised  £5million. The removal of duties on 600 goods, and the reduction on 500 more, considerably reduced the cost of living for the majority of the British population. It was also a step towards free trade, an issue which Peel had been greatly influenced by a free trade philosopher, Adam Smith, who argued the fewer restrictions, the better the economy would become. Peel also passed reform in 1844 with the Bank Charter Act creating a more efficient banking system on which the government could trust and rely, as well as the Companies Act which monitored companys progress and improved the nations financial health. Some historians have accused Peel about being too focused on his financial policies, however throughout his ministry he made genuine attempts to improve the conditions for workers, especially in mines and factories. Peel had always shown sympathy for the less-well off, and even early in his career in 1829 he had shown this through some of his reforms. His government set up of the National Relief Fund in 1841 to investigate the conditions for workers in growing industrial cities in addition to the pressure from leading figures like Lord Ashley, which prompted social reform. The reforms he made were not really radical, but were common sense and made the working environment for workers safer, reducing fatalities where death was frequent. The 1842 Mines Act forbade women and children under 10 to work underground, and the 1844 Factory Act which was largely driven by Peels home secretary, Sir James Graham, limited the hours children under 13 could work, and recommended safety improvements. The reform was successful in improving working conditions for some, and it reduced the death rates at work places as environments became safer. However, this did come at a cost in that it frustrated many Tory supporters as the efficiency of their industry was being compromised. Peels ministry also introduced The Railway Act in 1844 aimed to regulate the activities of railways to safeguard passengers interests, and called for a parliamentary train to run once a day which stopped at every station on the line. The improvement in living conditions for most people, and therefore success of Peels ministry, was marked by the decline of radical groups such as the chartists. One historian, Donald Read said Peel was: The hero of equally the newly enfranchised, the middle classes and of the unenfranchised property less masses. Peels ministry really benefited the poor and the middle class, as it narrowed the gap in some way between them, and the monopolising landowners through the introduction of fairer policies. The popularity of such groups like chartists depended on economic crisis which Peel effectively contained through timely and accommodating reform which kept the majority happy. Peel vitally saw that although it was important to maintain traditional Tory values to retain the support of much of his party, they were holding the party back in many ways, most of them unfairly and unnecessary. He worked around this by successfully diluting traditional policies to appeal to a wider number of people. Peels political career was closely intertwined with Ireland and OConnells career, an Irish political figurehead. Peels policies towards Ireland were on a whole relatively unsuccessful, and his attempts to win over moderate Catholic opinion while retaining the key features of the established church greatly failed, in some cases actually angering both Protestants and Catholics. The Irish Colleges Bill in 1844 which Peel hoped would bridge gaps between the Protestants and Catholics in Ireland ironically did the opposite. However, Peels policies to Ireland were hindered by Protestant Peers, and the self-interested landowners constantly slowing and refusing most of it, in the case of the Maynooth Grant in 1845, although it won over some catholic opinion it was passed too late, and just increased tension within the Conservative Party between Tory Ultras, and Peel. Despite Peels overall disappointment on the issue of Ireland, in 1843 he crucially took firm action to prevent the Repeal of the Union Act 1800, which made Ireland part of Britain. OConnell tried to use mass agitation to force the repeal as it had worked for him in 1829. However, when Peel recognised the seriousness of the threat from the Repeal Organisation as it became known, he made it clear he would be prepared to use force to uphold the act. Peel also took more decisive action in 1843 by passing a Coercion Act which banned OConnells huge meeting at Clontarf, and arresting him for conspiracy. This approach was successful in keeping Ireland in the Union, one of the many problems Peels ministry faced, although it did come at some cost as Irish nationalists now rejected OConnells constitutional approach and resulted to violence to bring about change. Peel has been named a betrayer of his party by some historians such as the modern historian Eric Evans who said Peel: Either ignored his followers sensibilities or bludgeoned them into submission. However, as Prime Minister of Britain he was using his skills as a leader and administrator not only to represent and benefit a small select group, but fairly to the whole nation. A successful ministry has to primarily address its nation and not its partys interests; this is what Peels ministry did. Although Peel had the backing of many Ultra Tories to get into government, Peel had very much created a new conservative party that stated they would reform if and when they saw necessary. In 1846 Peel saw the Corn Laws as the last major obstacle of achieving free trade. He believed that by repealing the Corn Laws British industry would become more competitive, and exports would be increased. He knew that he risked the unity of his part, and his own career but he believed that agricultural protection was no longer necessary or fair. They were in place for political and not economic reasons, and despite the Conservative partys negative view towards reform, Peel believed it would boost manufacturing, and lower prices for living, as well as demonstrate he was in touch with the needs of ordinary people. Peel did ultimately split the party he had created by forcing through the Repeal of the Corn Laws in 1846 however, he did it in the nations best interest, Britains economic prosperity for the next 30 years bearing witness to this. Some may argue that by splitting his party and not abiding to polices he was elected on, most notably he had promised not to repeal the Corn Laws, Peels ministry was a failure. He caused such a split in the party that it took around 30years for the conservative party to really find its feet again. Peels policy to winning over moderate opinion had been hugely successful nationally except it had come at a cost. Peels ministry from 1841 can be considered a success despite it concluding in the split of the Conservative party in 1846. This is because on a whole, the nation benefited greatly from moderate reform which caused working conditions to improve and the cost of living to be reduced, which improved living conditions. A greater deal of economic stability was reached as well as the national debt being eradicated. The reduction in radical activity emphasised the fact that conditions were improving in Britain at this time. As the average man of Britain benefited from Peels ministry, it came at a cost to the landowners and Tory Ultras who were a considerable part of the Conservative partys support base. In a sense Peels ministry was unsuccessful in terms of the negative impact it had on the Conservative party, and the tension that it created which peaked in 1846 as the Corn Laws were repealed and the Conservative party split. However, Peels ministry was prevailingly successful, in comparison to its party failure, as it greatly benefited the nation as a whole, most importantly though its financial reform, and also reaching its climax in 1846 with the Repeal of the Corn Laws.

Salvador Dali and Pablo Picasso Term Paper Example | Topics and Well Written Essays - 2750 words

Salvador Dali and Pablo Picasso - Term Paper Example The essay "Salvador Dali and Pablo Picasso" discovers the Salvador Dali's and Pablo Picasso's art. Both Salvador Dali and Pablo Picasso are well-known Spanish painters. They have not only accomplished a number of masterpieces that gained worldwide acceptance but they also received considerable criticisms that make them more famous. Dali, whose works usually deal with surrealism, is noted for absurdity. In his painting, Soft Construction with Boiled Beans, Dali presents an image of a monster tearing itself apart. This oil painting is usually referred to as a premonition of the Spanish Civil War because Dali made it six months before the war broke out. The painting, considered to be a surrealist painting, measures 100 cm x 99cm., and is currently housed in Philadelphia Art Museum of Art. Dali is famous for surrealism in his works. Convincingly the surreal nature of Dali’s images has led him to fame, but according to Saladyga, his â€Å"eccentric manner and attention-grabbing pu blic actions sometimes drew more attention than his artwork† (15). Specifically, Dali’s Soft Construction with Boiled Beans has gained him both positive and negative commentaries. Nevertheless, the subject, style, and symbols that he used make the painting a real masterpiece as it evokes the cultural and historical experience of the Spanish. Pablo Picasso is likewise a world-renowned Spanish painter and is even more famous and respected than Dali in the visual art scene. He is well-known for cubism, a style he himself made popular. With his craftsmanship and hard work, Picasso can be considered the most prolific artist not only of his time but even of all times. Throughout his lifetime, he produced a total of 6,000 paintings, 165 of which he painted in 1969 alone, when he was 88 years old (Whitman 53). He started painting at two years old and produced sketches, sculptures, lithography, and even plays before he died at 92 (Whitman 53). Guernica is one of his masterpieces . This oil painting, which measures 11  feet tall and 25.6 feet wide, was painted in 1937, two days after the bombing of Guernica. To date, it reminds the Spanish of the bombing of the Basque town of Guernica by some German bombers who were allegedly supporters of General Franco during the Spanish Civil War. At present, the painting is housed in Museo Reina Sofia, Madrid but a number of replications including the one in the United Nations have been on display worldwide. Both paintings mentioned reflect the painters’ view of the Spanish Civil War. According to Beevor (19), the war started on July 17, 1936 and lasted until April 1, 1939. It began after the proclamation of opposition by some Spanish generals led by Jose Sanjurjo against the leadership of the then President Manuel Azana. The group of generals were supported by a number of groups, including the Spanish Confederation of the Autonomous Right, Carlists, and Fascist Falange, which were strong political parties at th e time. Hence, the move resulted in political and military warfare. Later, the government obtained support from Germany, Italy and Portugal, thus heightening the conflict especially with the bombing of Guernica, Basque Country on April 26, 1937 by German and Italian troops. Guernica is said to be a marketplace, thus many people were hurt during the bombing, especially since that day was a market day.

Comparison & Contrast on Romanticism and Realism literary movements Essay

Comparison & Contrast on Romanticism and Realism literary movements - Essay Example Consequently, as industrial revolution progressed, these poor working conditions raised a lot of concerns and people started to advocate for changes. Artists and writers were amongst the people who were championing for the changes (Dahiya 4). They responded to these conditions in many different ways and as a result, romanticism and realism artistic movements emerged. Romanticism movement originated from Europe in the mid of the 18th century and was concerned with emotions and imagination (Morris, 169). It opposed aristocratic social and political ideologies of the age of enlightenment and the scientific rationalization of nature (Mendelowitz 153). Romanticism was highly centered on visual arts, literature and music but had greater impacts education and natural history (Morris, 49). During the 18th century, romanticism was common and popular (Reynolds 81). However, in the second half of 19th century it lost its popularity and was overtaken by realism movement. Realism movement was bas ed on real life and unlike their immediate predecessors (romantics), the practitioners of realism sought to portray real life, both good and bad. They were far more interested in science and in civilization, and far less interested in nature and the occult. Realists, like Romantics, were very moral writers, but moral causes changed with the times as new conditions dictated new issues (Richardson 56). Both romanticism and realism gathered a lot of fame during the era of industrial revolution. However, it was realism that was more popular from mid 1800s on. Romanticism only dominated from mid 1700s to the mid 1800s (Dahiya 4). Romanticism artists and writers put passion and emotions in their work. The works created by these writers and artists often dealt with themes that bring hope and inspiration or reflected nature’s beauty (Ferber 15). The works were always based on nationalism, spirituality and mankind. The works of the romantic artists gave the people an escape from the p roblems and challenges that they faced during the industrial revolution (Reynolds 111). The works of literature that are usually associated with this movement are the poems of William Wordsworth and William Blake. Other works include Mary Shelley’s novel, Frankenstein, which was a story about a scientist who tried to create human but created a monster, Alexandre Dumas’s work, The Three Musketeers and Victor Hugo’s novel, The Hunchback of Notre Dame (Richardson 67). The works of realists were based on real life. The art and literature developed or made by realists exposed life as it was. They clearly pointed out the difficult living and working conditions that were eminent during the era of industrial revolution. Most of the writers and artists of realism were activists who used their works to reveal the evils of society in the hope that they would be reformed. Realists had hopes that their works would bring inspiration to the politicians and other people in powe r thus enable them make lasting changes in living and working conditions (Herman 11). The realists were also referred to as social critics since they used to criticize the society. In US, the realists were referred to as muckrakers since they raked the muck that was in the society. The criticism of the realist was always on specific cause such as child labor, living conditions and working conditions in industries. Realists’ literature writers created factious works but their plots were real. The most known realist author is Charles dickens who was a British

Political Economy Lectures Essay Example | Topics and Well Written Essays - 750 words

Political Economy Lectures - Essay Example The Council on American Islamic Relations (CAIR) is an organization whose main aim is to advocate for the rights of Muslims in North America. It can be said that it represents all Muslim who live in North America. Its headquarters are situated in Washington D.C, and it has offices in at least twenty states and in Canada, and advocating for mutual understanding and justice is its vision. Its mission is to empower all American Muslims,, protect their rights, encourage dialogue and promoting understanding and justice through building coalitions. The talk informed us of what the Council on American-Islam Relations (CAIR) does in its day to day operations. They include, protecting the rights of American Muslims during questioning by the Federal Bureau of Investigations (FBI) and other law enforcement agencies, fighting for the rights of Muslims who have been subjected to employment discrimination and even those who have been terminated wrongfully. It also stopped unlawful seizers of prope rty by law enforcement and also advocates for equal student rights in schools and other learning institutions. The talks also sort to educate us on how people stereotype Muslims in America. ... Most people picked the first two photographs because the people in the photographs had dressed in a manner suggesting they were Muslims. The speaker showed how people were wrong by telling them that even the blond lady in one of the photographs was indeed a Muslim. The talk also informed us on the demography associated with Muslims in America. The speaker pointed out that Democrat accepted Muslim Americans more than Republicans. For instance the speaker pointed out that in the 2008 presidential elections seventy nine percent of Muslims living in America voted for the candidate from the Democratic Party. It was also pointed out that the Republican Party should advocate for the rights of American Muslim since they hold a significant percentage of the national vote. The theme of the talk was generally tolerance towards Muslims in America. The speaker aimed at showing how and why Muslims living in America are subjected to a lot of torment due to the issue of terrorism. He showed this by identifying the rise of Anti-Islam movements and Islam phobia in the United States of America. Islamophobia is an extreme fear of and hatred of Muslims. After the terrorist attack on the twin towers in September 11th 2001, hatred and fear of Muslim in America rose. Since then, Muslims in America have been put to discrimination and violence. ` After the talk, it was clear how Muslims in America have been oppressed. The talk made me realize that the opinion I had about Muslims in general was just a stereotype and that it was wrong. I came to understand what Muslims go through and how their rights are violated. I would encourage the Council on American-Islam Relation (CAIR) to continue with their good work in lobbying for the rights of

Plant DCL Proteins

Plant DCL Proteins Abstract Dicer, a double-stranded RNA (dsRNA)-specific endoribonuclease, plays an essential role in triggering both transcriptional and post-transcriptional gene silencing in eukaryotes by cleaving dsRNAs or single-stranded RNAs bearing stem-loop structures such as microRNA precursor transcripts into 21- to 24-nt small RNAs. Unlike animals, plants have evolved to utilize at least four Dicer-like (DCL) proteins. Extensive genetic studies have revealed that each DCL protein participates in a specific gene silencing pathway, with some redundancy. However, a mechanistic understanding of how the specific action of each DCL protein is regulated in its respective pathway is still in its infancy due to the limited number of biochemical studies on plant DCL proteins. In this review, we summarize and discuss the biochemical properties of plant DCL proteins revealed by studies using highly purified recombinant proteins, crude extracts, and immunoprecipitates. With help from co-factor proteins and an ATP ase/DExH-box RNA-helicase domain, the microRNA-producing enzyme DCL1 recognizes bulges and terminal loop structures in its substrate transcripts to ensure accurate and efficient processing. DCL4 prefers long dsRNA substrates and requires the dsRNA-binding protein DRB4 for its activity. The short-dsRNA preference of DCL3 is well suited for short-RNA transcription and subsequent dsRNA formation by coupling between a plant-specific DNA-dependent RNA-polymerase IV and RNA-dependent RNA-polymerase 2 in the transcriptional gene silencing pathway. Inorganic phosphate also seems to play a role in differential regulation of DCL3 and DCL4 activities. Further development of biochemical approaches will be necessary for better understanding of how plant DCL proteins are fine-tuned in each small RNA biogenesis pathway under various physiological conditions. Introduction RNA silencing, also known as RNA interference (RNAi), is one of the fundamental molecular mechanisms conserved in most eukaryotes to regulate gene expression both transcriptionally and post-transcriptionally. In both situations, what triggers the RNA silencing pathway is a small RNA molecule, 21 to 24 nt in length, called small interfering RNA (siRNA) or microRNA (miRNA) depending on its origin and the downstream pathways involved. The class 3 endoribonuclease (RNase) III enzymes known as Dicer are responsible for producing siRNA from longer double-stranded RNAs (dsRNAs) and miRNA from single-stranded RNAs with internal stem-loop structures   by a dsRNA-specific endoribonuclease. Therefore, the activity and regulation of Dicer-family proteins in a cell are vital to many biological processes requiring flexible adjustments at the level of gene expression, such as development, organogenesis, the circadian rhythm, biotic and abiotic stress responses, and defense against viruses an d transposons. Biochemical characterization of Dicers in animals The Dicer family is a unique class of RNase III enzymes due to the presence of an ATPase/DExD/H-box helicase domain at the N-terminus, a Piwi/Argonaute/Zwille (PAZ) domain in the middle and dual RNase III domains followed by one or two dsRNA-binding domains in the C-terminal half (exception: Giardia intestinalis) (Figure 1) (Bernstein et al. 2001). In general, the helicase domain serves as a protein-protein interaction surface recruiting co-factor regulatory proteins (Lee et al. 2006; Ma et al. 2008; Ye et al. 2007). It also utilizes ATP hydrolysis to achieve processive cleavage of the long dsRNA substrate (Cenik et al. 2011; Welker et al. 2010). The PAZ domain contains a conserved pocket for recognizing the terminus of the dsRNA substrate, and the distance between PAZ and the RNase III catalytic center determines the product sizes (MacRae et al. 2007; MacRae et al. 2006). Each of the two RNase III domains cuts one of the dsRNA strands, leaving a characteristic 2-nt overhang at 3-end of the product (Elbashir et al. 2001; Takeshita et al. 2007; Zhang et al. 2004). The C-terminal dsRNA-binding domains (dsRBDs) serve as a protein-protein interaction interface and nuclear localization signals, in addition to having dsRNA-binding function (Doyle et al. 2013; Hiraguri et al. 2005; Wostenberg et al. 2012). The specific functionality of each domain differs depending on the Dicer protein. Since the first demonstration of in vitro small RNA-producing activity of Dicer in the fruit fly Drosophila melanogaster (Bernstein et al. 2001), its biochemical properties and regulatory machinery have been extensively studied in humans, D. melanogaster and Caenorhabditis elegans. In humans, there is only one Dicer-family protein (hDicer), which cleaves short-hairpin pre-miRNAs produced by Drosha and dsRNA substrates into 20- to 22-nt small RNAs in an ATP-independent manner (Myers et al. 2003; Provost et al. 2002; Zhang et al. 2002). The cleavage activity requires a divalent metal cation such as Mg2+, Co2+ or Mn2+, and recognizes mainly the 5-end of the substrate to dictate the product length (Park et al. 2011). This 5-counting rule is reliant on the conserved 3-pocket motif within the PAZ domain and the 5-pocket motif, which is less conserved in Dicers of other eukaryotes. The binding of Dicer to a dsRNA substrate and its cleavage are uncoupled, because Dicer can bind to dsRNA with out Mg2+ or under low temperature (Provost et al. 2002; Zhang et al. 2002). The helicase domain of hDicer has an autoinhibitory function (Ma et al. 2008). In line with this, the activity of recombinant full-length hDicer protein can be improved under limited proteolytic conditions (Zhang et al. 2002). hDicer is responsible for both siRNA and miRNA production, and co-factor dsRNA-binding proteins TRBP and PACT dictate hDicer function in the two distinct small RNA production pathways (Chendrimada et al. 2005; Haase et al. 2005; Kok et al. 2007; Lee et al. 2013; Lee et al. 2006). In particular, the hDicer complex containing PACT disfavors siRNA precursor dsRNA and shows different cleavage patterns on the same pre-miRNA substrate than the hDicer-TRBP complex (Lee et al. 2013). The interaction with TRBP occurs through the hDicer helicase domain, and stimulates the hDicers catalytic activity. (Ma et al. 2008). Similarly, it has been reported that the C. elegans Dcr-1 interacts with a dsRNA-binding protein RDE-4 which enhances the Dicer activity toward long dsRNA substrates in siRNA production, while RDE-4 is apparently dispensable in miRNA production pathway (Parker et al. 2006; Parker et al. 2008; Tabara et al. 2002). D. melanogaster has two Dicer proteins, Dcr-1 and Dcr-2, which produce miRNA and siRNA, respectively (Lee et al. 2004; Miyoshi et al. 2010). Dcr-1 alone can process dsRNA into siRNA in vitro, but its interaction with the dsRNA-binding protein Loquacious isoform PB (Loqs-PB) confers pre-miRNA substrate specificity to the Dcr-1-Loqs complex by suppressing cleavage of long perfect dsRNAs and enhancing pre-miRNA processing activity (Saito et al. 2005; Zhou et al. 2009). Dcr-2 interacts with Loqs isoform PD and another dsRNA-binding protein, R2D2, in the siRNA production pathway (Liu et al. 2003; Liu et al. 2006; Miyoshi et al. 2010; Zhou et al. 2009). Dcr-2 alone is also capable of cleaving a pre-miRNA precursor in an ATP-independent manner, but R2D2 significantly suppresses Dcr-2 activity toward pre-miRNA, whereas Loqs-PD enhances the cleavage activity of Dcr-2 toward long perfect dsRNA precursors by boosting its affinity to the substrate (Cenik et al. 2011; Miyoshi et al. 2010). The pr ocessive processing of long dsRNA substrates by Dcr-2 depends on ATP hydrolysis by its ATPase/helicase domain, implying that one of the functions of the helicase domain is to allow Dcr-2 to produce multiple siRNAs from a single long dsRNA molecule before it dissociates from the substrate (Cenik et al. 2011). Such differential regulation of Dicer activity through specific interaction with co-factor dsRNA-binding proteins in distinct pathways is commonly found in most of the systems studied, including plants. DCL proteins in plants Plant genomes contain at least four distinct classes of DCL family proteins (DCL1-4). Like their animal counterparts, each class of DCL has evolved to participate in its primary pathway (Figure 2), but the three siRNA-producing DCLs (DCL2-4) function redundantly as well, because defects in one class of DCL can be compensated for by other classes in some cases (Gasciolli et al. 2005; Mukherjee et al. 2013; Xie et al. 2004). Because DCL1 is the only Dicer protein that produces most 21-nt miRNAs (Kurihara and Watanabe 2004; Reinhart et al. 2002), knockout mutants of DCL1 are embryonic lethal (Schauer et al. 2002). DCL4 is the major producer of 21-nt antiviral siRNA and endogenous siRNAs such as trans-acting siRNA and phased siRNAs (phasiRNA) (Bouche et al. 2006; Gasciolli et al. 2005; Mukherjee et al. 2013; Qu et al. 2008; Xie et al. 2005; Yoshikawa et al. 2005). DCL2 can compensate for the loss of DCL4 (Bouche et al. 2006; Gasciolli et al. 2005; Parent et al. 2015), although its major function remains unclear. DCL3 mainly produces 24-nt repeat-associated siRNAs derived from transposons and DNA repetitive elements, and participates in transcriptional gene silencing (TGS) through RNA-dependent DNA methylation, suppressing proliferation of these elements (Henderson et al. 2006; Pontes et al. 2006; Xie et al. 2004). In addition to the four classes of DCLs, monocots have another distinct class of Dicer, DCL5 (also known as DCL3b) (Margis et al. 2006). DCL5 is specifically expressed in developing panicles and is responsible for 24-nt reproductive phasiRNAs, although the biological significance of a reproductive-organ-specific 24-nt phasiRNA pathway mediated by this specific Dicer remains to be elucidated (Borges and Martienssen 2015; Fei et al. 2013; Kapoor et al. 2008; Song et al. 2012). This pathway might be analogous to the Dicer-independent PIWI-interacting RNA (piRNA) pathway in vertebrates, which suppresses transposons and other genes specifically in germlines (H irakata and Siomi 2016). Both forward and reverse genetics and physiological studies have successfully dissected the major RNA silencing pathways and allowed identification of the function of DCL genes in each pathway in plants. However, investigations on the molecular and enzymatic characteristics underlying the functional diversification and specificity of the DCL proteins are still in their infancy. Detection of DCL activity in crude extracts of various plants Biochemical characterization of plant Dicer activity was first demonstrated in wheat germ extract (monocot) and cauliflower extract (dicot), which contain multiple DCL activities producing ~21 nt and ~24 nt small RNAs with 2-nt 3-overhangs in the double-stranded form (Tang et al. 2003). These activities are weaker in the absence of ATP, consistent with characteristics of Dicer family proteins from Drosophila and C. elegans. Long dsRNA competitors effectively suppress both activities in wheat germ extract. The 24-nt small RNA producing activity was inhibited by 25-nt synthetic siRNA duplexes, whereas 21-nt small RNA production was unaffected by 21-nt synthetic siRNA duplex competitors, suggesting that two different enzymes with active sites that have distinct size-dependent binding properties are in the wheat germ extract (Tang et al. 2003). A recent study on wheat germ extract characterized these activities in further detail, revealing (1) that the 21-nt activity could be found in a much larger (~950 kDa) complex than the 24-nt activity, which had maximum activity in an approximately 450 kDa complex; and (2) the biochemical properties associated with the activities, such as divalent cation and NTP requirements, optimum NaCl concentration, temperature, and pH, and substrate length dependence (Shivaprasad et al. 2015). The identities of the DCL enzymes responsible for these activities in the wheat germ extract remain to be identified. A better understanding of the biochemical characteristics of individual plant Dicer proteins has come from the model plant Arabidopsis thaliana, which has four DCL proteins: DCL1, DCL2, DCL3 and DCL4 (summarized in Table 1). The first in vitro DCL activity in A. thaliana was demonstrated using a suspension cell lysate, a crude extract of inflorescence tissue, and an immunoaffinity-purified protein complex (Qi et al. 2005). Similar to the previous study using wheat germ extract or cauliflower, extracts from both Arabidopsis cultured cells and inflorescence tissue contained DCL dsRNA-cleaving activity producing 21- and 24-nt small RNAs from 400-bp dsRNA (Qi et al. 2005). The 21-nt producing activity and 24-nt producing activity were found in >660 kDa and ~400 kDa fractions, respectively, suggesting that these Dicers reside in protein complexes composed of multiple co-factors (Qi et al. 2005). In agreement with previous genetic studies showing CARPEL FACTORY/DCL1 is responsible for 21-nt miRNA production in vivo (Kurihara and Watanabe 2004; Reinhart et al. 2002), the 21-nt small RNA producing activity was DCL1 immunoaffinity-purified from inflorescence-derived crude extract by an anti-DCL1 antibody (Qi et al. 2005). The 24-nt activity was associated with anti-DCL3 antibody immunoprecipitate, and the activity was abolished when purified from a dcl3-1 mutant, showing that DCL3 is responsible for the 24-nt activity in Arabidopsis inflorescence extract. The immunoaffinity-purified DCL1 activity required ATP, whereas the activity of the DCL3 immunoprecipitate was ATP-independent (Qi et al. 2005). Interestingly, the dcl1-7 mutation did not abolish the 21-nt small RNA producing activity in the extract or immunoprecipitates, implying that the substitution (P415S) in its N-terminal helicase domain did not alter the enzymes catalytic activity itself (Qi et al. 2005); this study also found that the activity of DCL4 responsible for formation of 21-nt siRNA was present in the inflorescence extract. The presence of DCL4 activity in an Arabidopsis crude extract was demonstrated in later studies using 2-week-old seedlings as the starting material (Fukudome et al. 2011; Nagano et al. 2014), and will be discussed later in this review. In-depth biochemical characterization of DCL1, a microRNA-producing enzyme in plants  DCL1 activity requires DRB1/HYL1 and SERRATE for accurate processing of the miRNA precursor Both in wheat germ and Arabidopsis extracts, DCL activities are associated with size fractions larger than DCL monomeric form, implying that these DCLs form functional protein complexes composed of multiple co-factors in vivo. As summarized in an earlier section, such interactions between a Dicer and a co-factor protein are commonly found in mammals, nematodes and insects. One of the most characterized classes of co-factor proteins is a dsRNA-binding protein (dsRBP) harboring multiple dsRNA-binding domains or motifs. The A. thaliana genome encodes five dsRNA-binding (DRB) family proteins: DRB1/HYL1, DRB2 DRB3, DRB4, and DRB5. Multiple genetic and biochemical studies have demonstrated two specific interactions between DCLs and DRBs in A. thaliana: DCL1-DRB1/HYL1 and DCL4-DRB4 (Han et al. 2004; Hiraguri et al. 2005; Kurihara et al. 2006; Nakazawa et al. 2007). Arabidopsis DCL1, DRB1/HYL1, and another co-factor, SERRATE (SE), constitute an essential microRNA production pathway in vivo (Han et al. 2004; Lobbes et al. 2006). Unlike animals, which utilize two distinct RNase III enzymes, Drosha and Dicer, for the first and second cleavage of microRNA precursors, plants do not employ Drosha. Therefore, the DCL1-complex is responsible for the processing of both primary and precursor miRNA substrates. The detailed molecular machinery of the dual miRNA processing mediated by DCL1 and the co-factor proteins have been extensively studied biochemically using highly purified recombinant proteins produced in heterologous systems (summarized in Figure 3). One of the systems utilizes baculovirus-mediated recombinant protein production in Sf21 insect cells, followed by two-step affinity purification (Dong et al. 2008). The highly purified recombinant DCL1 protein alone could process a 94-bp dsRNA substrate with a 2-nt 3-overhang into 21-nt small RNA in an ATP/Mg2+ dependent manner. The optimum NaCl concentration for the activity was 25-50 mM, and a NaCl concentration higher than 100 mM severely impaired the activity (Dong et al. 2008). While the recombinant DCL1 protein alone could produce 21-nt small RNA from both primary and precursor miRNA (pri-/pre-miR167b) substrates in vitro, DRB1/HYL1 and SE recombinant proteins co-incubated in the same reaction mixtures significantly increased both yield and accuracy of the processing (Dong et al. 2008). Without these co-factors, more than 80% of 21-nt small RNA products from the DCL1-alone reaction were due to incorrect processing from the end of the primary miRNA substrate, whereas the processing mediated by the DCL1-DRB1/HYL1-SE complex produced accurate 21-nt products with a sequence identical to miR167b/miR167b*, amounting for up to 81% of the products (Dong et al. 2008). This demonstrated that accurate processing of miRNA precursors by DCL1 requires the co-factors DRB1/HYL1 and SE. Cons istent with a previous study, the interaction between DCL1-DRB1/HYL1 through the second dsRNA-binding motif of DCL1 is important for the precise processing of pri-miRNA in A. thaliana (Dong et al. 2008; Kurihara et al. 2006). Also, using highly purified recombinant proteins and surface plasmon resonance analysis, it has been suggested that DCL1 changes its structural conformation when it binds RNA and exposes more binding sites for SE (Iwata et al. 2013). Binding to substrate dsRNA or miRNA precursors might be an important regulatory step for DCL1 dicing activity, as its dsRNA-binding domains exhibit the strongest binding to dsRNA among the four Arabidopsis DCLs (Hiraguri et al. 2005). ATPase/DExH-box RNA-helicase domain of DCL1 suppresses its dicing activity, confers ATP dependence, and influences processing accuracy In addition to its RNase III and dsRNA-binding domains, the helicase domain of DCL1 plays a significant role in regulating its dicing activity. Two independent forward genetic studies have identified two dcl1 mutant alleles, dcl1-13 (E395K) and dcl1-20 (R363K), as hyl1 suppressors, and the amino acid substitutions of both alleles occur within the ATPase/DExH-box RNA-helicase domain. These dcl1 mutations partially rescue the accumulation of some miRNAs in a hyl1-2 mutant (Liu et al. 2012; Tagami et al. 2009), and dcl1-13 was at least partially able to restore the phenotypic defects of hyl1-2 such as a reduced number of rosette leaves and a   leaf shape (Tagami et al. 2009). Highly purified recombinant DCL1-20 protein exhibited enhanced catalytic activity (Kcat/Km) toward pri-miRNA156a compared to wild-type DCL1 (Liu et al. 2012). Similarly, the helicase domain-deleted DCL1 recombinant protein (DCL1à ¢Ã‹â€ Ã¢â‚¬  Helicase) showed higher processing activity in vitro and was no l onger dependent on ATP for its activity toward pri-miRNA156a (Liu et al. 2012), suggesting that the helicase domain of DCL1 might have an autoinhibitory function like that of human Dicer (Ma et al. 2008; Provost et al. 2002). The in vivo miRNA processing imprecision in hyl1-2, however, was not restored by a dcl1-20 mutation, implying that the partial recovery of the hyl1-2 mutant, including miRNA accumulation, was due to the enhanced catalytic activity resulting from the substitution in the helicase domain (Liu et al. 2012). Interestingly, the effect and magnitude of DRB1/HYL1 and DCL1 helicase domain seem to vary among miRNA precursors. For example, the in vivo processing accuracy of miR156a is much more severely affected by hyl1-2 mutation than miR166b is (Liu et al. 2012). pri-miR156a is processed from the loop-proximal site to the loop-distal base in vitro (Liu et al. 2012), which is considered unusual for plant miRNAs (Addo-Quaye et al. 2009; Mateos et al. 2010). Accurate processing of pri-miRNA166b by native DCL1 is largely dependent on the presence of ATP, and processing by DCL1à ¢Ã‹â€ Ã¢â‚¬  Helicase is less accurate than that of native DCL1 (Liu et al. 2012). In contrast to miR156a, the processi ng precision of which is markedly affected by hyl1-2, that of miR166b was much more impaired by dcl1-20 mutation than hyl1-2 (Liu et al. 2012). Also, the effect of the other helicase mutant allele, dcl1-13, on miRNA production was shown to depend on the presence or absence of DRB1/HYL1 in vivo (Tagami et al. 2009). These observations indicate that efficient processing of different miRNA precursors by DCL1 have different reliance upon DRB1/HYL1 and DCL1helicase domain that potentially depends on structural determinants of the miRNA precursors. Structural determinants for efficient and accurate processing of miRNA precursors by DCL1 Primary transcripts of miRNA (pri-miRNA) have a characteristic secondary structure: a loop-distal stem (lower stem), a miRNA duplex, a loop-proximal stem (upper stem) and a terminal loop (Figure 3). Typical miRNA maturation from these precursors requires at least two cleavages occurring at the lower and upper stems. In animals, the single-stranded base region of the loop-distal stem is recognized by the dsRNA-binding protein DGCR8, which guides the processing center of Drosha to the correct position, which is 11 nt from the base of the stem (Han et al. 2006). However, this distance-from-base rule is not sufficient for plants because the length of the loop-distal stem of plant pri-miRNAs is highly variable (Song et al. 2010). Several structural features of pri-miRNAs that influence the activity, binding position and directionality of the processing by DCL1 have been elucidated genetically and biochemically (Figure 3a, b). One structural determinant lies within the loop-distal stem of pri-miRNA. For the first cleavage at the loop-distal stem, bulges and unpaired regions play a major role in the efficiency of miRNA processing. Mutant pri-miRNAs with closed bulges were processed at the correct position, but resulted in the accumulation of unprocessed pre-miRNAs in vivo, indicating that the rate of subsequent processing at the loop-proximal stem was impaired (Song et al. 2010). In pri-miR171a, which has a long loop-distal stem, the first cleavage position was determined by the distance from a relatively unstructured region instead of the base of the stem; the conserved distance from an unstructured region of the lower stem important for miRNA processing was found to be approximately 15 nt (Figure 3a) (Mateos et al. 2010; Song et al. 2010; Werner et al. 2010). The 15-nt rule was essentially reproduced in an in vitro miRNA processing system using highly purified DCL1-DRB1/HYL1-SE recombinant proteins and an artificial pri-miRNA substrate bearing another unstructured region in the elongated lower stem. In addition to the canonical processing, another type of processing occurred at 15 nt from the artificially introduced unstructured region, validating the functionality of the 15-nt rule (Song et al. 2010). The importance of bulges and unpaired regions in the lower stem for processing by DCL1 might explain why some miRNAs with a near-perfect matched stem seem to be DCL4-dependent, rather than DCL1-dependent (Rajagopalan et al. 2006; Song et al. 2010). On the loop-proximal and terminal loop side, a branched terminal loop (BTL) or a large terminal loop was found to be an essential structural factor that may alter directionality of processing by DCL1 and the resultant miRNA-accumulation (Figure 3b). BTL induces abortive processing of pri-miR166c both in vivo and in vitro (Zhu et al. 2013), meaning the first cleavage of the pri-miRNA occurs in the loop-proximal stem as opposed to the normal productive processing beginning in the loop-distal stem. The molecular basis of this bidirectional processing by DCL1 was further investigated using an in vitro system that reconstitutes the DCL1-processing machinery. For this purpose, DCL1, DRB1/HYL1 and SE harboring Agrobacterium tumefaciens were co-infiltrated to Nicotiana benthamiana leaves, and the transiently expressed DCL1-DRB1/HYL1-SE complex was immunoaffinity-purified two days after infiltration (Zhu et al. 2013). The reconstituted DCL1 complex cleaves the substrate pri-miRNA 16-17 nt fro m the unpaired region of the lower stem, supporting previous studies (Mateos et al. 2010; Song et al. 2010; Werner et al. 2010). By disrupting one of the two RNase III domains of DCL1 alternately and using 5- or 3-end labeled pri-miR166c substrates, the bidirectional nature of both productive and abortive processing was demonstrated (Zhu et al. 2013; Figure 3b). The helicase domain of DCL1 fine-tunes the position of both productive and abortive processing by DCL1 in an ATP-dependent manner (Zhu et al. 2013). DCL1à ¢Ã‹â€ Ã¢â‚¬  Helicase complex could not abortively process a substrate with BTL. Also, wild-type DCL1 required ATP for abortive processing, but not productive processing, indicating that the ATPase-driven helicase activity is necessary in abortive processing to unwind the structured BTL (Zhu et al. 2013; Figure 3b). In productive processing, the effect of helicase deletion and ATP depletion depend on the distance between the processing site and the bulge in the lower stem. Many potential byproducts of the abortive processing of pri-miRNA precursors with BTL can be found in publically available high-throughput small RNA sequencing data from both Arabidopsis and rice, implying that both substrate structure and the functionality of the ATPase/helicase domain of DCL1 are conserved mechanisms to regulate miRNA biogenesis in higher plan ts (Zhu et al. 2013). Dissecting distinct characteristics of DCL3 and DCL4 activities DCL4 activity requires the dsRNA-binding protein DRB4 In A. thaliana, DCL2, DCL3 and DCL4 are responsible for producing various siRNAs 21-24 nt in length. The dsRNA-cleaving activities of DCL3 and DCL4 can be detected in crude extracts prepared from 2-week-old seedlings (Fukudome et al. 2011). Extracts from wild-type seedlings cleave 500-bp dsRNA substrates into 21-nt and 24-nt small RNAs. In this system, the 21-nt and 24-nt small RNA-producing activities can be attributed to DCL4 and DCL3 respectively, because the dsRNA-cleaving activity of the corresponding size was abolished in each of the single mutants (Fukudome et al. 2011). Also, a mutation in the dsRNA-binding protein DRB4, which interacts with DCL4 (Hiraguri et al. 2005; Nakazawa et al. 2007), abolished DCL4 activity in seedling extracts. The DCL4 activity could be further purified by immunoprecipitation with anti-DCL4 or anti-DRB4 antibodies. The immunoaffinity-purified DCL4 requires Mg2+ and ATP for its activity, and is inhibited by >200 mM NaCl. This property is similar to t hat of recombinant DCL1 protein (Dong et al. 2008). The DCL4 complex immunoprecipitated from the drb4-1 mutant did not show dsRNA-cleaving activity, but the addition of recombinant DRB4 protein to the complex restored the 21-nt producing activity in vitro, showing that DRB4 functions as an essential co-factor for the dsRNA-cleaving activity of DCL4 (Figure 4b). In this system, mutant DRB4 proteins harboring substitutions in the conserved amino acid residues that form a hydrogen bond with the phosphodiester backbone of dsRNA at the dsRNA-binding site (H32A in the first dsRBD and K133A in the second dsRBD of DRB4) lost their ability to interact with dsRNA and DCL4, and did not restore DCL4 activity. The second substitution (K133A) alone impaired its interaction with the C-terminal half of DCL4 containing two RNase III domains and two dsRBDs in a GST pull-down assay using recombinant proteins, but was not sufficient to block restoration of DCL4 activity when added to DCL4 immunopurified from a drb4-1 mutant extract. There might be an add itional interaction surface between DCL4 and DRB4 involving dsRBD1 of DRB4 and the N-terminal half of DCL4, which contains an ATPase/DExH-box RNA-helicase domain and an RNA-binding domain (formerly known as domain of unknown function DUF283; Figure 1), as their specific interaction was reported in vitro (Qin et al. 2010). Short dsRNA preference of DCL3 activity orchestrates 24-nt siRNA biogenesis in TGS pathway Crude extracts from 2-week-old seedlings have also been used to characterize substrate specificity of DCL3 and DCL4. Consistent with the long dsRNA preference of Drosophila Dcr1 (Bernstein et al. 2001), DCL4 preferentially cleaves longer dsRNA substrates, and is less efficient in producing 21-nt siRNAs when the substrate is shorter than 50 nt (Nagano et al. 2014). On the other hand, DCL3 activity, producing 24-nt siRNAs, favors shorter substrates such as 30 nt and 37 nt dsRNA with a 1-nt or 2-nt 3-overhang (Nagano et al. 2014). It also favors substrate dsRNA with 5-adenosine or uridine. The 24-nt small RNA produced by DCL3 has a 2-nt 3-overhang, and the cleavage follows the 5-counting rule proposed for human Dicer (Park et al. 2011). DCL3 is not reliant on ATP hydrolysis for activity, as it can still process the short dsRNA substrate in the presence of a non-hydrolyzable ATP analog, adenosine 5-O-(3-thio)triphosphate (Nagano et al. 2014). Unlike DCL4, which targets long dsRNAs such a s RDR6-dependent TAS dsRNAs or exogenous viral dsRNAs in vivo (Bouche et al. 2006; Dunoyer et al. 2005; Qu et al. 2008; Yoshikawa et al. 2005), DCL3 may not need to perform a processive cleavage, which requires ATP hydrolysis, because the length of its targets allows only a single cut (Figure 4a). The DCL3 preference for short dsRNA substrate is consistent with the one precursor, one siRNA model for RNA polymerase IV (Pol IV)-dependent 24-nt siRNA biogenesis (Blevins et al. 2015; Zhai et al. 2015). In this model, a remarkably short (30- to 40-nt) transcript with 5-adenosine is produced by Pol IV and is simultaneously converted into double-stranded form by an RNA-dependent RNA polymerase, RDR2. The short dsRNA substrate is processed into 24-nt siRNA preferentially by DCL3 due to its length specificity, facilitating the subsequent RNA-directed DNA methylation process (Blevins et al. 2015; Zhai et al. 2015). The transcription of short RNAs by Pol IV, and the length and 5-adenosine substrate preference of DCL3 might be essential mechanisms to prevent other DCLs from processing specific dsRNA substrate needed for the TGS pathway. Such coupling of RDR-Dicer-RNAi is also known in fission yeast, where a Dicer physically interacts with an RNA-dependent RNA polymerase to form coupled ma chinery that drives siRNA-mediated TGS (Colmenares et al. 2007). In addition, DCL3 can participate in 24-nt siRNA production from longer transcripts with aid from another RNase III enzyme, RNase III-like 2 (RTL2). As a class II RNase III enzyme, RTL2 possesses one RNase III domain and two dsRBDs, and is involved in rRNA maturation [in vivo is implied]in A. thaliana (Comella et al. 2008). Recombinant RTL2 protein can cleave long dsRNA substrates into 25 bp or longer dsRNA in vitro (Kiyota et al. 2011). Recently, it has been shown that RTL2 processes a subset of Pol IV-dependent dsRNA into shorter intermediates, which are preferable for DCL3 activity in vivo (Elvira-Matelot et al. 2016). Although no direct interaction has been reported, RTL2 and DCL3 can be considered other examples of coordinated action of a dsRBD-containing protein and a Dicer in plants. DCL3 is also reported to physically interact with the dsRNA-binding protein DRB3 in the antiviral RNA-directed DNA methylation pathway (Raja et al. 2014). The function of DRB3 in DCL3 activity rem ains elusive. Inorganic phosphate, NaCl and KCl differentially regulate DCL3 and DCL4 activities In the same assay system using crude extracts, inorganic phosphate at a physiological concentration promotes DCL3 activity but suppresses DCL4 activity toward 50-nt dsRNA substrates (Nagano et al. 2014). The differential effe

The impact of Queen Victorias Death on Australia :: essays research papers fc

Queen Victoria’s Death Queen Victoria’s reign, lasted 63 years, from 1837 to 1901. She ascended the throne of Great Britain, when she was 18. Despite being the Queen of Britain, and a very influential and prominent person, she also had an impact on Australian history. In Australia the most apparent legacy of Queen Victoria’s reign is manifested in the names of the two states, Victoria and Queensland. There are also a large number of other important buildings and places, named in her honor such as, the Queen Victoria Building, in Sydney. Victoria was loved and respected by her subjects, and Australian society at the time was affected greatly by the loss of their beloved Queen. An example of the affection and grief felt by the Australian people is apparent in the following poem, written by J D Horne of Castlemaine, Victoria. â€Å"Who can recall to memory the life Of one so loved, so dear — A devoted mother, Queen and wife A friend when few drew near — And not in silent agony Be melted into tears. A nation weeps her, The whole world mourns her... Dead, did I say? Ah! No! She lives In every loving heart. A nation’s grief is not assuaged – No time can heal the smart. She lives forever, and her deeds Will live in memory too, And history recount her praise For all the ages through. For never monarch lived on earth So worthy of our song...†   Ã‚  Ã‚  Ã‚  Ã‚  The Victorian era, as it became known, was the time of Queen Victoria’s reign. Victoria’s death signaled the end of an era. It was a rich and significant period in Britain, which had a positive influence on Australia also. It saw profound social changes culminating in the rise of the middle class, population growth, and an increase in wealth. Victoria was Queen and so it was seen as a product of her reign and so the love for her increased. Victoria’s death created a feeling of insecurity and uncertainty of what would result. However, it also created the feeling of opportunity and a new beginning. â€Å"There was a sense not only in London but in all the capitals of the world that an age had come to an end, a stronger sense than there had been when the 19th century itself drew to a close.† (1).   Ã‚  Ã‚  Ã‚  Ã‚   There were mixed feelings with the end of Victoria’s reign. At the time Australian culture still revolved around Britain and most Australian’s were either born in Britain, or had relatives residing there.