Asda Strategic Management Essay

I.Introduction Asda was first formed in 1965 by a group of Yorkshire farmers and originally sold only grocery products. However in 1990 Asda sold its first range of clothing under the name ‘George’ and was soon in most of their stores. In 1999 Wal-Martbought Asda and has since moved into other retail areas such as electronics, home appliances and home, travel and motor insurance, as well as setting up an order and delivery service (Saunders, 2011). Now Asda is the third-biggest supermarket in the UK with over 500 stores across the UK and provide around 35,000 products to customer. The following report will illustrate business-strategy level of Asda by using some strategic analysis model like PEST, SWOT, Porter’s Five Force and Porter’s Generic Strategies.  This report also gives to Asda some advices for future development strategy. II.Content 1.The wider environment in the UK. PEST analysis is a simple tool, useful and widely used.It will help to capture the â€Å"overview† of political, economic, cultural – social and technological environment in the UK Political The political environment is ideal for business with a stable friendly government. The political environment in the UK is considered to be relatively stable. The UK is a nation under a constitutional monarchy. Head of state is the king, but the king only symbolic role. Executive power is exercised on behalf of the king, but in the hands of actual government. The head of government is the Prime Minister. The Prime Minister is the leader of the majority party in the House of Representatives seat (Williams, 1998). Generally the British political system little changed. Even the legal system of the State policy also does not change much Biggest concern of businesses is the continuity of the rules and principles of conduct whether any of the parties are in power. A change in government, whether by election or coup not always mean changing the level of political risk. Contrary to the fundamental change in policy could create major instability. A sudden change in unpredictable government policies can also hinder business activities of the enterprise, regardless of the changing cause.Soï ¼Å'for political factor, in the future years and current, Britain basic political situation is stable. This situation is good for British supermarket industry to grow in strength. Read more:  ASDA Jobs Economic In the early 20th century, the global economy has made profound changes due to the volatility of the political situation in the world. The world economy achieved high growth rates in the 2000s and began to decline sharply after terrorism of 9/11 in America (Colin Grahame Bamford, 2002). The volatile world economy has profoundly affected European countries such as the U.S., Japan †¦ but the UK economy remains sustainable. UK also is an attractive market for foreign investors because the UK has many advantages, such as the financial centre of the world currency, infrastructure development, and tax is lower than the EU other, skilled workforce with relatively low labour  costs compared with developed countries. Currently, the UK is attracting foreign investment (FDI) in most EU regions. In 2008, the UK accounted for 30 % FDI in the EU, accounting for 9.3 % of world FDI. As predicted, in 10 localities FDI only leading the world in 2001-2005, England was ranked 2nd after receiving the U.S. with an average amount of FDI per year is estimated at 82.5 billion dollars. UK is also investing abroad largest EU. In 1998-2005, an annual average of UK investment abroad amounted to U.S. $ 119.4 billion, private investment in 2008 totalled nearly $ 250 billion (ONS, 2012) . The more important is the UK is The UK is the fifth largest economy country in the world, the second largest exporter and the third largest importer of commercial services, eighth largest exporter and fifth largest importer of merchandise. Social In 2007, the UK population was reached 61 million.UK is one of most densely populated countries. Growing of UK population isabout nearly 400,000 one year. Population growth tended to influence retail trade. More and more potential customers will be faced by ASDA in the future. The increasing population can give ASDA good opportunity for making more profit. Technology Science and technology is developed in the UK now.By 2008 in the UK,there is about 16 million households use the internet for buying food. This was UK households 65%. (Na 2008) 2.The competitive environment within the UK. Analysis of the Porter’s five forces for the purpose of establishing the position of a company in that industry. The factors in the model of Porter is: The five forces are threat of substitute products, bargaining power of buyers, bargaining power of sellers, competitor rivalry and threat of new entrants Future developments in the industry and the ability of the company to respond. The threat of substitute products The threat of substitute products is very low for ASDA. It is the product convenient hiding in the corner or the license can be considered as the replacement product. However, because the less of the product at the corner store so customers will still have to go to the supermarket to meet their shopping needs Bargaining power of buyers ASDA is the largest distributor of influence throughout the UK, the system of the ASDA distribution can affect many industries such as food, electronic goods, commodities and consumer goods daily. ASDA has enough power to negotiate with other companies on price, product quality and marketing policies to put products into your system. Threat of new entrants It can be said pressure of ASDA from competitors is very low. Because open a create a new supermarket or start to run a new business to enter the market you have to reach two conditions, which are invest a huge amount of capital and huge supply system to compete with ASDA. Bargaining power of suppliers The provider can afford to have bargaining power pay the price in a few cases when only a few suppliers. Bargaining power of suppliers is very low for ASDA when they supply goods under the terms were drafted. ASDA had concerns about branding and enhance the reputation, such as set up ASDA Foundation in 1988 to assist any charity in the UK, and it is benefit for brand construct and is to be a leader that enhances customer loyalty. And more and more buyers want to go shopping at ASDA so the supplier power will increase in this situation. Finally, ASDA’s productions are more and more competitive advantages. Existing rivalry between competitors The competition between existing competitors is very high because the supermarket industry in the UK is saturation. Therefore the competition within the industry appears to be intense. ASDA is affronting many internal and external competitors such as TESCO, SAINSBURY,MORRISON and AHOLD of the Holland. TESCO is the largest retailer in the UK. It can contend for markets with ASDA by its large number stores and attractive price. SAINSSBURY is the second one in UK, its productions famous for high quality. While AHOLD is the largest supermarket in Holland, therefore it has the absolute strategies. All of these have a high degree to share market with ASDA. 3.Asda’s strategic position within the UK. SWOT analysis will be the useful tool to point out the strategic position of Asda within the UK industry Strength Strengths of ASDA Group is a system consisting of many stores spread across the UK. Chain stores have proven to be an ASDA strong brand. Now, the corporations in approximately 136 company retail stores. ASDA pursue low pricing strategy. It aims to offer a shopping basket which is 10—15% lower than the chief competitors. ASDA considered the method that can use the Consistent low prices rather than advertisement to give the confidence to customer. It is that does not need to buy some products in other supermarket for pursue the value price. In addition, growing market share is strength, ASDA is the second largest food retailer in the UK, ASDA has outperformed the rest of the retail sector in terms of growth rate and continues to gain market share as shoppers respond to its reputation for low prices. Weakness Since this is a target retailers with cheap so some time in the company sold inferior quality products and this significantly affected the reputation of the company. The main weakness of ASDA is its lack of small and midsize supermarkets and small convenience stores in the UK, it is clearly that many European countries like France and Germany has places hard restrictions on construction of big stores in suburb and rural areas. Although most ASDA supermarkets fall into big style, but ASDA’s biggest rival has two-third of outlets are small or midsize supermarkets. (Matlack, 2005) states that â€Å"Even if ASDA big supermarkets are doing well, it is difficult to expand with that format.† Opportunities Also for large scale so the management and coordination of management activities is very difficult. There are many opportunities that can be exploited ASDA, which is expanding its market to potential markets such as the EU. In addition the company can exploit some other business sectors such as tourism, insurance. ASDA have many opportunities in many fields. Firstly, with the process of economic globalization, many emerging markets are growing fast, like the turnover of China retail industry is increasing about 30% for every year. ASDA can access to the new market in the future.  Secondly, it can grow organic food market. Rising health awareness among consumers led to a higher demand for low calorie organic foods around the world. Customers are becoming aware of the risks associated with obesity and poor dietary habits. ASDA has taken many initiatives to cater to the health-conscious consumer. Threats Supermarket business is highly profitable industry, many investors want to invest. Currently in the UK there are several large supermarkets are competing with Tesco and Morrison’s ASDA. Initially, the UK retail industry is highly consolidated with Tesco and Sainsbury. Tesco is key competitor of ASDA. It operates through the multiple store formats, including Extra, Superstore, Metro, Express and hypermarkets. Besides in the UK, Tesco also operates in other European countries and Asia. Furthermore, the labour wages go up in the UK, according to the Department of Business Enterprise and Regulatory Reform, labour costs are raising in the UK. It would adversely influence ASDA’s margins. 4.The competitive strategy of Asda. For a big retailer as Asda, a sustainable competitive advantage is the key to get success. A sustainable competitive advantage is about performing different activities or performing similar activities in different ways. That means, Asda must be capable of producing value for the consumer that is recognized as being superior to that of its competitors. The best ways to achieve this goal is that Asda should follow one of three generic competitive strategies which developed by Porter. Figure 1: Porter’s Generic Strategies The first strategy is overall cost leadership, this strategy requires Asda provide lowest-cost products to their consumers without reduce quality of products. Overall cost leadership allows Asada to outperform rivals within the industry because it can charge lower price and its lowest-cost base still allows them to earn profit. The second strategy is differentiation, if Asda use this strategy, Asda must provide different goods and services which  satisfy the needs of their customers. Differentiation requires Asda spend more cost to create and keep their competitive advantage. However, this cost can be offset in case Asda can increase revenue. The third strategy is focus, Asda can focus on cost leadership or differentiation in a narrow market. Asda can become the cost leader but only within a specific market or Asda can offer special products, service with high value within a segment of market. In pursue focus strategy, Asda could develop their brand loyalty which help Asda decrease threat of new entry. One of dangerous problem in applying Porter’s Generic Strategy is that some organizations try to do all three and become stuck in the middle. However, in case of Asda it not appropriate, right from the first days of establishment, the company has put out a formula for their business is cost reduction to always discount sales. That means Asda choose cost leadership strategy to achieve competitive advantage in the supermarket industry in UK. To following this strategy, Asda always guarantee give to their customer 10% cheaper than their competitor (Asda, 2014). Besides that, Asda also try to minimize their staff’s number by using shopping online and self-service check out to reduce cost. Online shopping will reduce retailer costs because less staffs are needed in shops to serve the customer. The annual report in 2009 show that self-service checkouts are becoming more favoured in Asda store and the wage costs was saved  £10 million. In addition, Asda also try to increase their economics of scale to reduce production cost in long term. In 2010, Asda paid  £778 million to take over the Netto – this is an aggressive bid to close the gap with market leader Tesco’ (The Guardian 2010). This action is evidence shows that Asda using economics of scale pursue cost leadership strategy. Another action shows the cost leadership strategy of Asda pay a large cost for their own machines and the result is that in the long term production of their own brands will be cheaper than other. Another measure of Asda in reducing costs is hard bargaining strategy with their suppliers. It means that they decrease power of suppliers, if their current suppliers do not meet their requirement they can simply move to another supplier that will meet their demands. More and more Asda is choosing to use suppliers from abroad because they are often cheaper than suppliers in Britain and regulation is less strict meaning they are able to achieve their cost leadership strategy. (The Guardian) 5.Future development strategy of Asda. Asda level strategy Asda has many stores around UK, but the company not expand around the world. The firm must develop and try to stay in the top of sales in the UK and try to compete with international companies. Asda now is the second largest retails in the UK by market share. Strategy of Asda, Asda will base on two strategies. First one internal growth and the second one is acquisition. First direction is internal growth, This direction will help the company to develop the products by using internal resources. Asda will open new electronic firm called Asda electronic, and this firm will produce some home appliances. This will help Asda to have more profit and to become the first largest retails in the UK by market share. This means the company will pay a lot of money in the beginning to build the new firm, and in the research but after that the revenue will rise. Second direction is alliances, It’s so important for any firms to extend to another country, but in the same time it’s difficult. The strategy for Asda is to alliances of another company like Aldi. Aldi is German company and has over 8,000 stores worldwide, the company opened in 18 countries around the world. And that’s very important for Asda to become multinational firm. This alliancewill help the firm to make more profit. Ansoff matrix, Figure 2: Ansoff matrix Asda will development new products to the market in the UK. And the firm will produce home appliances like cooker and microwave and all the home machine. New market, Asda plan to expand in different countries by alliances with Aldi. That will raise the revenue, and help the firm to become multinational. Criteria suggested Suitability The strategies suitable for Asda, these strategies will move Asda from local company to multinational company. Because the firm will alliances another company Aldi. This means the number of customers will increase, and the profit will also rise. Feasibility Now is the best time to do these strategies, Asda make huge profit in the last few years. This makes it easy to Asda to build the electronic firm and to acquisitions. Acceptability The strategies are acceptable to the stakeholders of the business, because it will raise the profit of the company. And in the same time they will not do loss money. III.Recommendation Based on strategy of view of us, Asda should choose market development like a future strategy. In more specific, Asda should do alliance with Aldi because of two reasons. Firstly, the market for supermarket in the UK does not have lot space to develop, so expand to international will give to Asda more profit. Secondly, Aldi is giant company with wide range of store on the word; alliance with Aldi not only helps Asda expand to Germany but also helpsthe firm will come multinational. All that will make the firm become bigger and have change to get stronger in the UK and capture the market share in other countries. In addition the firm will receive a huge profit as boost the revenue, build anextremelystrong brand image. IV.Reference 1.Na, 2008 People Prices Planet [online] Available at: http://www.about-asda.com/inside-asda/people-prices-planet.asp (Accessed 20/03/2014) 2.TRAVIS, LAN (2008) Boom in births brings UK population to almost 61m [online] Available at http://www.theguardian.com/world/2008/aug/22/population.immigrationandpublicservices (Accessed 10/04/2014) 3.Carol Matlack 2005 Wal-Mart’s Overseas Stumbles [online] Available at: http://www.businessweek.com/bwdaily/dnflash/jul2005/nf20050727_3355_db016.htm (Accessed 05/04/2014) 4.The Guardian (2009), Zoe Wood. Asda suppliers under pressure[online]. Available at http://www.guardian.co.uk/business/2009/mar/22/asda-supermarkets (Accessed 05/04/2014) 5.The Guardian (2008), Finch. J. Asda wants ethical code for UK suppliers only[online]. Available at http://www.guardian.co.uk/business/2008/apr/07/asda.supermarkets (Accessed 30/01/2014) 6.Porter, M. (1980), Porters (1980) Generic Strategies, Performance and Risk, pp 8 7.ASDA. (2012). Asda Official Webpage. Available at http://www.asda.co.uk (Accessed 13/05/2014) 8.Abraham, C. S. (2007). Strategic Planning: A practical Guide for Competitiveness Success. Bingley: Emerald Group Publishing: 9.Colin Grahame Bamford, S. G. (2002). The UK Economy in a Global Context. London: Heinemann Education Publisher. 10.Kotler, P. (2007). A framework for Marketing Management. Upper Saddle River, New Jersey: Pearson Prentice Hall. 11.Mike Storry, P. C. (1997). British Cultural Identities. London. 12.ONS. (2012). Office for National Statistic. Available at http://www.ons.gov.uk/ons/index.html (Accessed 07/04/2014) 13.Porter, M. E. (1980). Competitive Strategy: Techniques for Analyzing Industries and Competitors. New York: Free Press.

Application of Colloids

Applications of Colloids| Colloids play a very important role in nature, in our daily life and in industry. Some of the important applications of colloids are discussed below. 1. Food stuffs and medicines:  Many of our food stuffs are colloidal in nature. Milk, butter, whipped cream, fruit jellies, ice cream, bread etc. are all colloidal in nature. For example, milk is an emulsion of butter fat in water, stabilized by milk protein (casein). Ice cream is a dispersion of colloidal ice particles in cream. Similarly, bread consists of air dispersed in baked dough.Colloidal medicines are more effective and are easily absorbed by the body system. Therefore a large number of pharmaceutical preparations are emulsions. Halibut-liver oil, cod-liver oil, skin ointments etc. are emulsions. Antibiotics such as penicillin, streptomycin etc. are usually injected in the body in colloidal form. Several metal sols are also used as medicines. 2. Purification of water:  In water works, water is usua lly purified by the addition of certain electrolytes such as potash alum, aluminium sulphate etc. This involves the phenomenon of coagulation.The impure water usually contains dispersed colloidal particles which cannot be removed by filtration. When potash alum is added to impure water, the negatively charged colloidal particles of impurities get coagulated by the action of Al3+  ions furnished by the alum and can be removed by filtration or decantation. 3. Sewage disposal:  Sewage water contains particles of dirt, mud etc. which are colloidal in nature and carry some electrical charge. These particles may be removed by using the phenomenon of electrophoresis.The sewage water is passed through a tunnel fitted with metallic electrodes and maintained at a high potential difference. The colloidal particles present in the sewage water migrate to the oppositely charged electrodes and get coagulated. This solves the problem of sewage disposal. Moreover, the rubbish matter obtained on account of the coagulation of colloidal dirt particles may be used as manure. | 4. Smoke precipitation:  Smoke is colloidal system and consists of electrically charged colloidal particles of carbon dispersed in air. As smoke is a big source of pollution.It is always desirable to precipitate it, i. e. , to remove colloidal carbon particles present in it. The removal of colloidal carbon particles from smoke can be effected by using the phenomenon of electrophoresis. This is achieved in an apparatus called Cottrell precipitator as shown in figure. Smoke is allowed to pass through a chamber having a number of metal plates attached to a metal wire connected to a source of high potential (20,000 to 70,000V). The electrically charged colloidal particles of carbon and dust get discharged when come in contact with the oppositely charged plates and fall down to the bottom.The clean hot air leaves the precipitator from an exit near the top. 5. Artificial rain:  Clouds are colloidal systems and consist of water vapour mixed with dust particles. The water molecules present in a cloud develop some electrical charge. Therefore, clouds can be made to rain by neutralizing the charge present on colloidal particles (water molecules). This type of rain is called artificial rain and may be carried out by spraying oppositely charged colloidal dust or sand particles over a cloud. This neutralizes the charge on water molecules and compels them to get coagulated, i. . to rain. 6. Rubber industry:  Latex obtained from rubber trees is an emulsion consisting of a dispersion of negatively charged rubber particles in water. In order to obtain rubber from latex, the latter is boiled when rubber particles get coagulated. The coagulated mass is vulcanized and sold as natural rubber. Rubber-plated articles can be prepared directly from latex by electrically depositing the negatively charged rubber particles over the article to be rubber-plated by making the article an anode in the rubber -plating bath. 7.Leather tanning:  During the tanning of leather, the positively charged colloidal particles of raw skin and hides are coagulated by the negatively charged tanning materials which include tannin and compounds of aluminium and chromium. On soaking hides in the solutions of tanning materials, the coagulation of negatively charged tanning materials takes place in the pores of hides and the latter get hardened. 8. Cleansing action of soaps:  Soaps solution is colloidal in nature and removes dirt and oil from clothes by forming water soluble emulsion as explained earlier. . Smoke screen:  In chemical warfare, smoke screens are generally used to hide the movement of troops. Smoke screens are the colloidal dispersions of very fine particles of titanium oxide in air. 10. Formation of delta:  When a river falls into the sea, extensive deposits of sand and clay are formed at the mouth of the river in the sea. Such deposits are called delta. The river water contains col loidal particles of sand and clay which carry negative charge. The sea water contains a number of positive ions such as Na+, Mg2+, Ca2+  ion etc.When the river water comes in contact with sea water, the negative charge present on colloidal sand and clay particles get neutralized by the positively charged ions (present in sea water) and they get coagulated. The coagulated sand and clay particles settle down and take the shape of delta in due course of time. 11. Blue colour of the sky:  The sky is the empty space around earth and as such has no colour. It appears blue due to the scattering of light by the colloidal dust particles present in air (Tyndall effect). 12. Preparation of Nano-materials:  Nano-materials which are used as catalyst are prepared by using reverse micelles. 3. Building roads:  Now a day, roads are built asphalt emulsified in water. This technique does not require the necessity of melting the asphalt. 14. Metallurgical operations:  Several metal ores are concentrated by froth floatation process. This process involves the treatment of the pulverized ore in emulsion of pine oil. What are the Applications of Colloids? Colloids play a very significant role in nature and in our daily life. Some of the important applications of colloids are discussed below: 1) Medicines: Medicines in colloidal form are easily adsorbed by the body tissues and hence are more effective. ) Sewage disposal: Colloidal particles of the dirt, mud etc. carry electric charge, hence when sewage water is passed through the plates kept at a high potential, the colloidal particles are coagulated due to electrophoresis and the suspended matter gets removed. 3) Purification of water: The precipitation of colloidal impurities present in water can be done by adding certain electrolytes like alum etc. the negatively charged colloidal particles of impurities get neutralized by the Al3+ ions and settle down and pure water can be decanted off. 4) Cleansing action of soap: Soap solution is colloidal in nature.It removes the dirt particles either by adsorption or by emulsifying the greasy matter sticking to the cloth. 5) Formation of Delta: River water contains charged colloidal particles of clay, sand and many other materials. Sea water is very big store house of a variety of electrolytes dissolved in it. As soon as river water comes in contact with sea water coagulate the suspended colloidal particles which ultimately settle down at the point of contact. Thus the level of the river bed rises. As a result, water adopts a different course and delta is formed in due course of time. ) Smoke precipitation: Smoke particles are actually electrically charged colloidal particles of carbon in the air. Precipitation of smoke particles is carried out by Cottrell precipitator which is based on the principle of electrophoresis. Smoke is allowed to pass through a chamber having a number of metal plates attached to a metal wire connected to a source of high potential. C harged particles of smoke get attracted by oppositely charge electrode get precipitated after losing their charge and the hot air passes out through the chimney.The dust particles are also removed in this process. Thus the nuisance of smoke in big industrial cities can be avoided. 7) Photography: A colloidal solution of silver bromide in gelatin is applied on glass plates or celluloid films or paper to from sensitive plates in photography. 8 ) Artificial rain: Artificial rain can be caused by spraying oppositely charged colloidal dust or sand particles over a cloud. The colloidal water particles present in the cloud will be neutralized and coagulate to from bigger water drops causing artificial rain. 9) Rubber industry:Latex is a colloidal solution of negatively charged rubber particles. From latex, rubber can be obtained by coagulation. Rubber plated articles are prepared by depositing negatively charged rubber particles over the article to be rubber plated by making that article a n anode in a rubber plating bath. 10) Smoke screen: In warfare smoke screens are used which are nothing but colloidal dispersion of certain substances in the air. 11) Other applications: a) Blue color of the sky is due to the scattering of light by colloidal dust particles in air (Tyndall effect).Similarly, sea water looks blue due to scattering of light by the colloidal impurities present in sea water. b) Tail of comets is seen as a Tyndall cone due to the scattering of light by the tiny solid particles left by the comet in its path. c) Blood is a colloidal solution and the stoppage of bleeding on applying ferric chloride solution is due to coagulation of blood forming a clot. Colloids-Chemistry and Applications What are Colloids ? In a true solution as sugar or salt in water, the solute particles are dispersed in the solvent as single molecules or ions.Thus the diameter of the dispersed particles ranges from 1A to 10 A. On the other hand, in a suspension as sand stirred into water , the dispersed particles are aggregates of millions of molecules. The diameter of these particles is of the order 2,000 A or more. Applications of Colloids Colloids play an important role in our daily life and industry. A knowledge of colloid chemistry is essential to understand some of the various natural phenomena around us. Colloids make up some of our modern products. A few of the important applications of colloids are listed below. (1) Foods:Many of our foods are colloidal in nature.Milk is an emulsion of butterfat in water protected by a protein, casein. Salad dressing, gelatin deserts, fruit jellies and whipped cream are other examples. Ice cream is a dispersion of ice in cream. Bread is a dispersion of air in baked dough. (2) Medicines: Colloidal medicines being finely divided, are more effective and are easily absorbed in our system. Halibut-liver oil and cod-liver that we take are, in fact, the emulsions of the respective oils in water. Many ointments for application to s kin consist of physiologically active components dissolved in oil and made into an emulsion with water.Antibiotics such as penicillin and streptomycin are produced in colloidal form suitable for injections. (3) Non-drip or thixotropic paints: All paints are colloidal dispersions of solid pigments in a liquid medium. The modern nondrip or thixotropic paints also contain long-chain polymers. At rest, the chains of molecules are coiled and entrap much dispersion medium. Thus the paint is a semisolid gel structure. When shearing stress is applied with a paint brush, the coiled molecules straighten and the entrapped medium is released. As soon as the brush is removed, the liquid paint reverts to the semisolid form.This renders the paint ‘non-drip’. (4) Electrical precipitation of smoke: The smoke coming from industrial plants is a colloidal dispersion of solid particles (carbon, arsenic compounds, cement dust) in air. It is a nuisance and pollutes the atmosphere. Therefore, before allowing the smoke to escape into air, it is treated by Cottrell Precipitator. The smoke is let past a series of sharp points charged to a high potential (20,000 to 70,000 V). The points discharge high velocity electrons that ionise molecules in air. Smoke particles adsorb these positive ions and become charged.The charged particles are attracted to the oppositely charged electrodes and get precipitated. The gases that leave the Cottrell precipitator are thus freed from smoke. In addition, valuable materials may be recovered from the precipitated smoke. For example, arsenic oxide is mainly recovered from the smelter smoke by this method. (5) Clarification of Municipal water: The municipal water obtained from natural sources often contains colloidal particles. The process of coagulation is used to remove these. The sol particles carry a negative charge.When aluminium sulphate (alum) is added to water, a gelatinous precipitate of hydrated aluminium hydroxide (floc) is formed, A l3+ + 3H2O —- Al(OH)3 + 3H+ Al(OH)3 + 4H2O + H+ —– Al(OH)3(H2O)4 The positively charged floc attracts to it negative sol particles which are coagulated. The floc along with the suspended matter comes down, leaving the water clear. (6) Artificial Kidney machine:  The human kidneys purify the blood by dialysis through natural membranes. The toxic waste  products such as urea and uric acid pass through the membranes, while colloidal-sized particles of  blood proteins (haemoglobin) are retained.Kidney failure, therefore, leads to death due to accumulation  of poisonous waste products in blood . Now-a-days, the patient’s blood can be cleansed by shunting  it into an ‘artificial kidney machine’. Here the impure blood is made to pass through a series of  cellophane tubes surrounded by a washing solution in water. The toxic waste chemicals (urea, uric  acid) diffuse across the tube walls into the washing solution. The purified blood is returned to the  patient. The use of artificial kidney machine saves the life of thousands of persons each year.The phenomenon of the scattering of light by the sol particles is called Tyndall effect. (7) Blue colour of the sky:  This is an application of Tyndall effect. The upper atmosphere contains colloidal dust or ice  particles dispersed in air. As the sun rays enter the atmosphere these strike the colloidal  particles. The particles absorb sunlight and scatter light of blue colour (4600–5100A). The light that  Ã‚  is incident at earth’s surface is considerably reddened due to the removal of most of the blue light in  the upper atmosphere.

Prima facie Essay

Introduction According to the article, Rhino poaching is in no way shown as moral. The ethical issues I see are that people are ignoring the fact that this horrible act is occurring and many people who do know about it won’t do anything about it, but are able to waste time watching pointless videos. The You Tube interventions took a moral approach to help with the petition. Although some were offended, the majority signed the petition and became more aware of the world around them. Utilitarianism When studying the supreme principle of morality as utility, we must first examine the definition of utilitarianism. Utilitarianism the effort to answer the question of man ought to do. For a utilitarian, the answer is simple: Act to produce the best consequences possible for the greatest number of people possible. In this, liberty and harm are treated as an equal. The end goal is to produce a general welfare or Arthur’s collective well- being. Jeremy Bentham, one philosophical view we examined defined utilitarianism as the ethical system that judges actions to be moral to the extent they maximize happiness, producing pleasures, and preventing pains. According to Bentham, there is a possibility of good and bad consequences however; preventing suffering is what matters through pleasure and the avoidance of pain. John Stuart Mill was a follower of Benthams, and he came up with the principle of utility. He stated that â€Å"Nature has places mankind under the governance of two sovereign masters† these masters are pain and pleasure. This is an experience based principle. We learn through experience that we are governed through pleasure and pain. According to Brandt’s view on utilitarianism, if all you do is add up numbers, there still a possibility of producing an immoral outcome. Singer’s principles also exemplified this. In the article â€Å"YouTube Interventions to Save the Rhino†, Utilitarianism is exemplified in that there was a greater outcome for a greater number of people. Sure, some were offended, however in the cases that the petition was signed, the rhinos and animal activists were impacted positively. Also, the new act allowing this method of communication will help when it come t other disastrous situations. Also, the whole world was able to be impacted. This effort had a mass effect on the petition. In the end, rhinos could be saved and a great idea was introduced, even the offended learned that their time was not being used effectively and therefore had somewhat of a positive outcome. Deontology In the study of deontology, we use Kant and Foot’s philosophical views. Deontology can be considered duty-based ethics, and reason alone should be used when finding the moral duty this concerns and reason in turn will cause a respect for rationality. Kant believed that morally you should act so that the maxim of your actions can and should be considered a universal law; morally you should have respect for human dignity. In this principle one should never for any reason intentionally harm someone who is believed to be innocent. Philippa Foot expands of Kant’s principle of hypothetical imperatives in an argument. She argues the Kant contrasts acting out of respect for moral law with acting from an alterior motive. Taking this into consideration she believes is crucial to shape Kant’s moral Philosophy. All in all, morality can only involve rational beings because only rationa l beings have the capacity to reason the way things are and should be and the ability to exercise freedom. Perfect categorical duties allow for no if’s and’s or but’s. Others are not, under any circumstances, to be used just as a means to acquire morality. In summary of this moral standing and the three forms of hypothetical morality according to Foot and Kant, â€Å"If you want x, you should do y, Because you want x you should do y, and because x is in your best interest, you should do y†. For Kant the second and third principles are one in the same. The article answers the supreme moral question â€Å"Did anyone use anyone merely as a means? † The answer is simply yes. The multiple videos that were altered were used as a means to get the communication across about animal poaching. The You Tube interventions had a positive outcome however in that a mass majority was made aware of what was going on in the world and how much time the viewers were actually wasting watching the highly viewed frivolous viral videos. Deontologists would agree with the interventions campaign. They wanted people to be aware of the issue of rhino poaching and wanted a petition signed so they included a link to the petition and urged people to sign it by noting that it wouldn’t take much time. They also wanted people to be aware that the silly videos were simply a waste of time and that was noted. Deontologists would have resolved these issues in a very similar manner because no one was hurt, they were just helped. Prima Facie Duties The prima facie duties introduced by W. D. Ross, a professor from Oxford University, argued that the right and the good are properties known intuitively and these duties may conflict holding only prima facie. There are no supreme principles involved. All focal points in the argument of what makes right acts right and wrong acts wrong are taken into account when looking into prima facie duty. Prima facie is judgment based on considered opinion. The article doesn’t really exemplify prima facie duties in that the article agrees completely with the study of deontology, and Ross argues with deontology. The duty to sign the petition however did arise from the obligation to save the rhino’s from poaching. This exemplifies the opinion Ross had on duty in itself. Conclusion I thought that the approach this article took was completely effective and I agree that the awareness of rhino poaching was done morally. When others want to make you aware of things like the animal fur industry, they walk down runways with imitation blood dripping from a fur coat. This was done in a way where most were not offended, a majority of people were impacted and action was taken. The petition was signed and an increase of 400% of the signatures was reached. I think that deontology supports the article the most. The x and y principles were exemplified as Foot and Kant had demonstrated. I think that all of the principles we studied in this section can be applied to the article. The article least agrees with prima facie ideas however. There are ways to apply it which I stated earlier in this essay. So what makes right acts right and wrong acts wrong? How many are influenced, how they are influenced, and act to include intent of agent and consent of person affected by act.

Consumer Protection Essay Example | Topics and Well Written Essays - 2500 words

Consumer Protection - Essay Example There were also certain ambiguities related to the actual people accountable for the faulty transaction. Hence, the author stresses on the need for stringent laws to protect the consumers' interests in business transactions. In addition, there appears to be an urgent need for a watchdog which would ensure that all laws are adhered to, in turn facilitating fair business practises. In the language of ecology, consumers are defined as those organisms who survive by depending on others around them (Consumer, AbNature, n.d.). This dependence could be for any day to day necessity, which they themselves are incapable of producing. And there are instances when triggered by the acute need of the consumer, this dependence on the producer has transformed into a form of exploitation, thus giving a freehand to the producers to make a kill. This definition and example might seem a little far fetched at the moment, but surprisingly both are in a way applicable in our day to day lives. There appears to be no end to our needs - material or spiritual and there are providers for all our needs. From the time we wake up each morning till the time we go back to sleep, knowingly or unknowingly, willingly or unwillingly, all of us tend to depend on one fellow human being or other for various reasons - hence, in a sense of speaking we all are consumers. Till the time all transactions happen in good faith and to the satisfaction to all parties involved, the world is a dreamland! However, the problem arises when the transactions do not happen in the true sense of fair practises. And considering that there are so many transactions happening, the possibility of exploitation cannot be ruled out. So, if we agree that we have advertently or inadvertently involved ourselves in one business transaction or the other, there have been at least one or two instances in our lives when we have felt that we as consumers have been given an unfair deal by the providers/salesman and the deal has not been concluded in the manner it should have been; in other words, our rights as customers have been infringed. The deal could be anything ranging from sale of faulty products, refusal to replace faulty products, unsatisfactory post-sales maintenance to poor service. And most often than not, in spite of continuous follow ups we have failed to get our issues addressed or resolved and the inconvenience compensated. Consumer protection is the current buzz word worldwide. There has been an increased awareness about the enormity of this need and there has been an increase in awareness among customers about their rights. Policies are being formulated, traders practising their trade unfairly are being implicated and there is a worldwide movement towards ensuring that the consumers get their due in all business transactions. However, in the absence of clear directions, policies and guidance on the grievance redressal systems, it is not always that the consumer is protected from unfair business practises. b. Objectives The need for consumer protection persists worldwide and it is no

(managing human capital) Traditional performance appraisal methods and Essay

(managing human capital) Traditional performance appraisal methods and why it leads dysfunctional behaviour - Essay Example Performance appraisals are undertaken to assess the individual, a team, or an organisational unit's progress as far as its function is concerned. They can be based on personal assessment of the ratee based on a prepared rating scale depending on the systems preferred by the management. In the general context, performance evaluation is a good tool to reward and recognise outstanding performances and correct or counsel employees with poor accomplishments (US Department of the Interior, n. d) Traditional methods of performance appraisal methods may differ in usability between private and public organisations. But they have similarities as far as the rating systems are concerned. Old systems use review processes in which the supervisor writes his ratings and opinions of the performance of a subordinate based on a form. In some organisations, the staff member is asked to fill out a self-review to share with the supervisor. In most cases, the appraisal reflects what the rater can remember and not a comprehensive evaluation. It is based on feelings and can be judgmental in nature (Dulewicz, 1989) Considering that humans have emotions, traditional assessment techniques and even some present methods are mostly ceremonial rather than a thorough assessment of the personnel's o

Short story Essay Example | Topics and Well Written Essays - 750 words - 4

Short story - Essay Example Normally, two spoons are enough for me. However, that day I needed more. It was like I felt that that one extra spoon would give me the confidence and courage for the grand adventure that was just about to begin. That morning I was going to ride my bike to the top of the highest mountain in the neighborhood. The steep twisted roads leading to the top were considered dangerous even for the more experienced riders than I was. The challenge seemed to be a tough one. However, I had been crazy about reaching that summit for almost a year, and I had to do it. Otherwise, my desire would not leave me alone. I finished my super energetic drink and headed for the front door. Although my courtyard was lit with the sunlight, it was still quite chilly outside. The whiff of fresh air against my face cheered me up as I moved towards the mountain. I was as excited as never before. After I crossed the railroads, my challenge was officially on. When the first corner was complete, I found that my vigor had gone somewhere. The feeling was like myriads of invisible needles were stuck into my legs. Sweat was streaming down my face. I could feel how it gets into my eyes. It was like someone stuck a finger straight into my eye socket. I stopped to remove my glasses just for a moment; then went on again. As I turned around the fifth corner, I realized how close to the top I was. My heart was jumping out of the chest. The sound of it was ringing in my head like a bell; I could practically hear it pumping blood throughout my exhausted body. The summit was very close. I was nearly there. However, I was starting to flag. My legs moved slower and slower. It was harder to press the pedals. I felt like I was underwater. Climbing up on the last hill, I saw a beautiful glade through the trees. Obviously, it was my final leap. At that moment, it was rather a mental challenge than a physical one. That was the point when I had to question myself how much I wanted to reach the top. I

Pleas rewrite this essay about political economic

Pleas rewrite this about political economic - Essay Example In regards to the authors, their argument logic exemplifies that there is a link between colonialism and development (historical determinant), that areas where settlers from Europe have reached are prospectively to have potential for enhanced institutions. The authors had stated a theory that different kinds of policies of colonialism resulted to diverse kinds of institutions from settling states and extractive states, settlement feasibility influenced the theory of colonization in regards to the extractive state having unlivable environment, and colonial states and institutions continued after attaining independence. They supposed that the manifestation of settlers from Europe during the colonialism era led to a considerable influence on the development in the areas of their settlement. The authors also avail evidence regarding their argument concerning economic growth and historical determinants. For instance, Canada, the united States, as well as, Australia where Europeans immensely settled have representative and protective institutions. Thus, they argue that colonization or the European settlement served as a foundation for sturdy institutions and future growth, as well. Additionally, they demonstrated that minimal distortionary policies and enhanced secure property rights would aid in investing more in human and physical capital that will be greatly utilized to attain an advanced income level. Further, they also possess check and balances systems to make certain equal power among government branches. On the contrary, an extractive state such as Congo that has extracted from Belgium is characterized by the absence of check and balances and property rights to impose exploitation from the government, and their key aim emphasizing on transferring resources to Belg ium. This discourages opportunity from the development of the economy. Nonetheless, the argument by Glasser was a counter argument. Glasser posit

A project study for new entrant restaurant in West London, Northolt Essay

A project study for new entrant restaurant in West London, Northolt - Essay Example In case the findings will be encouraging, a thorough feasibility study should establish further details like the allowable amount of capitalization needed in order to be competitive, why such an amount will be right, how to raise the funds, and who will be the owners by the time the actual business becomes a reality. Thus, the initial scope of this project study will be a top view of the Restaurant Industry in London and West London, Horholt as well as an inside view of the factors affecting the industry as well as other factors that can positively or negatively affect a new entrant in the restaurant business Among others, the location itself ought to be described. And the restaurant which is in between an industrial estate, plans to penetrate the competitive market shortly after it was opened. How to gain a good share of the potential business for restaurants within Northolt will be the other major problem that this research should be able to resolve. In other words, the aim of this project is first of all to clarify whatever opportunities, limitations, and requirements have to be considered by a new industry player in the restaurant industry. Secondly, it aims to find out whether the present restaurant can have the capability to rise up to the minimum requirements in order to compete in promoting the business. II. Structure and Elaboration of the Process In order to determine the potential of a restaurant business in the area and later on recommend a market strategy for the purpose of gaining a good market share, a more vivid description of the critical parts of a restaurant industry would have to be presented. These would include the following: (a) Location - descriptions of the place where the restaurant is people; (2) People – or who should be served by any promotion and set of services plus other benefits; (3) Products – or what will have to be offered to the potential customers; (4) Pricing – comparison of the cost of products and se rvices offered in the restaurant; and (5) Current Promotions – how restaurants in Northolt promote their business in order to build a market share for themselves. It will be assumed that the new restaurant does not yet have such a strategic promotion to likewise reap a good market share. Potential of the Place The location of Northolt is considered â€Å"a pleasant suburb to the West of Greater London, in Middlesex† according to Squidoo (2012). It has parks, transportation facilities linked to the rest of London, Golf and Country Clubs, and a Royal Air Force airport. A traveller will take only 20-30 minutes coming from Central London to reach Northolt. One of the transport facilities is an underground Railway Station that is connected to the underground London Railway Station. The cost of rent or purchase price of a house was recently reported to be very reasonable. There are car repair shops, football fields, playgrounds for children, a park for skating, and a boat la ke. Squidoo calls the place â€Å"cosmopolitan and multicultural†. But it still has woodlands, lakes in parks, wildlife where a moorhen, geese, swans, and ducks. A Coffee Shop beside the lake has become popular. Near the Northolt Railway Station, there are more than 20 hotels. See the complete list in Figure 1 under the Appendix Section. This gives an idea of how many people visit, rent, and live in Northolt. The Ealing

My Bus Tour from South Dakota, Pierre and to Mount Rushmore Essay - 9

My Bus Tour from South Dakota, Pierre and to Mount Rushmore - Essay Example The bus trip set off from South Dakota. Driving along the main road in South Dakota graced the cruise with the beautiful scenery of the Mythic Mount Rushmore. Driving across South Dakota to its outskirts covered over 200 miles across what was nothing less than jaw-dropping and cool scenery. The day-long drive took us to Pierre. As the bus cruised in the main road, we ogled at the beautiful sight of the mountain ranges cascading over each other, throughout the entire stretch. The green leafy vegetation and forest overgrowth complemented the beauty of the mountains and serve as homes of reindeers which occasionally leaped out and into the road. We could not be any closer to nature and its captivating beauty. In instances where the manmade activities interfered with the end trail of natural vegetation, we cited an array of dining joints, small-scale and large-scale casino saloons and lodging which testified of the industriousness of the people of South Dakota. From Pierre, we followed the US Route 83 to get across the Missouri River. As we turned westwards, the striking beauty of grasslands that accompanied us to Interstate 90 appeared to dance beautifully against the gentle breeze. From this juncture, we turned off to Murdo for tantalizing buffalo burgers. From Murdo, we drove to the Badlands National Park. One of the most memorable encounters in Badlands National Park was the dusty canyons therein. The Rapid City for dead American presidents also clearly served as another feather in Badlands National Park’s cap. The images of the dead American presidents are carved into the Black Hills and its high aesthetic presentation of America’s four great men and founding fathers served as the trip’s denouement.  Ã‚  

Small-Business Preference Essay Example | Topics and Well Written Essays - 750 words

Small-Business Preference - Essay Example The Congress has managed to come up with some very important and exciting programs that regulate and guide use of unmanned aircrafts or rather drones. The United States law enforcing agencies have taken steps to incorporate use of domestic drones. This has greatly helped enforcing agencies improve in their surveillance capabilities within the American population and without the American population. It is important to note the fact that incorporating such drones in public life would definitely change the American public life as it is known. It is therefore wise to consider implication of unmanned aircraft presence and capabilities of these flying objects. The government must therefore be careful to ensure there is public support for these unmanned flying objects. Normally, being monitored and tracked through surveillance mechanism will cause some discomfort among the public and affect public life eventually. Because of such guidelines drone manufacturing is only limited to domestic us e in case of business related interests and not wars. Manufacturers of drones have brought forward several suggestions for consideration by the United States Homeland security department. Examples include arming the remote controlled unmanned aircrafts with weapons such as tasers tear gas or rubber bullets. Such use is limited to non lethal weapons by the law enforcement agencies. Several states have enacted laws that govern use of domestic drones offering insight to the kind of businesses that can be allowed for manufacture (New York Times, 2012). Usage limits for drones as legislated include only when warrants for their use have been issued, in emergency cases or when there is enough support to conclude its use will assist in collection of important evidence to specific criminal activities. When image collection capabilities are incorporated in the drones, such images should be saved or retained only when it will be useful as evidence. Policies guiding use of domestic drones inclu de having the public representatives and not police departments make such decisions. It also says that such policies should be clearly written and open for public use. Part of the guidelines also includes a public audit of its use and a properly instituted oversight committee to prevent misuse. Domestic weapon shouldn’t also carry lethal weapons for safety of the public in case of misuse by a rogue regime. Small-Business Programs Created By Congress and How They Benefit Remote Controlled Aircraft Manufacture Businesses Over Large Air Craft Multinational Organizations Examples of how unmanned and remote controlled aircraft have assisted law enforcement agencies include the documentation of crime scenes in western Colorado. Similar remote controlled aircraft have been used in places such as England, Kenya and South Africa to watch for poachers that are almost bringing wildlife conservation efforts to its knees. It’s also thought courier services might incorporate use of these drone to reduce business running costs by delivering mails. The movie industry will too get a boost by offering directors views only helicopters managed to capture. This is important for their work and in improving quality of their services and products (Congressional Research Service, 2012). The same kinds of planes have taken to the skies in South Africa to pursue suspected poachers seeking rhino horns. They have been tested in England and South Africa

Assisted Suicide Essay Example for Free

Assisted Suicide Essay Often labeled compassionate, according to the columnist for the Wall Street Journal, Joni Eareckson Tada, legalized assisted suicide or euthanasia is really a deadly double standard for those with severe disabilities, both conditions that are labeled terminal and those that are not. She seems to be of the opinion that euthanasia should never be considered an option no matter how severe or subjective suffering the person is facing. In the article, Tada claims that legalization of euthanasia, â€Å"sends a clear message that a person with a disability may not have a life worth living† (1). She touches on euthanasia in The Nethlands and Belguium . She stands for a powerful example of a person with this depilating condition, in this case quadriplegia, can accomplish in spite of her condition. However this is somewhat of a bias perspective because it implies that she has put a measure on the subjective pain this condition and similar to it entails. There should be certain conditions set in order to qualify for euthanasia so that it is an option for people who want it but we can also make a system to prevent people from misusing it. We were granted free will which includes the right to end our life when we chose. There does not need to be this all or nothing attitude towards assisted suicide and the disabled. Tada argues, â€Å"this ruling sends a clear message that a person with disability may not have a life worth living†(1). This law would bring nothing but a choice for the terminally ill. There are some disabilities that are incurable and cause tremendous suffering. They should not all fall under one umbrella. Andrew Btiva wrote in his article, Disability and physician-assisted suicide, â€Å"We do not believe that the right to assisted suicide is premised on a diminished quality of life for people with disabilities. It is based on respect for the autonomy of terminally ill individuals during their final days. It does not deny people with disabilities suicide prevention services, protection against murder, or protection from other abuses†(1). Tada is very bias in this article claiming that it would take away the disability will to live and go against their advocacy to have hope and live. When a person comes disabled and loses control in many ways, this in fact just gives them something that they can control. There are defiantly some factors that should qualify for a person requesting assisted suicide. They should be terminally ill with no hope of recovery and  in great physical pain. If it were legalized the disabled population would be majority of the prospect’s. I don’t believe that all disabled should qualify automatically as Tada argues in her article claiming that if legalized all disabled including children and obese would qualify for assisted suicide as an option. Tada states, â€Å"Society’s moral and unwritten law has always led us to save our children-and certainly not allow them to destroy themselves†(2). I do agree that children should ever be considered for assisted suicide unless they are in dire suffering with no hope of recovery. Tada argues, â€Å" a child lacks the mental, emotional and psychological maturity to make such a decision about death†(2). I completely agree with this but if you had your child suffering and in a vegetative state with no chance of recovery wouldn’t you want to put them out of their misery? We don’t let animals suffer so why let humans? My aunt had a very full life at 52 she found out that she had pancriaic cancer and after she went to the doctor twice she was informed that she had nothing more than IBS. I had advised her that she should get further checked out. And in June of 2010 she went to the doctor with horrible pain in her stomach and after many tests she was informed that she was diagnosed with stage 4 pancreatic cancer. At that point she was also told that she would not be able to get the surgery to remove it because it was at the lower end of the pancreas where it would be more difficult and could pass from the operation. She was told she could do Chemotherapy. After doing 6 month of intensive pain ridden sessions she realized that she didn’t want to do that any more. Even tho she did not want to end her life she did request that when she was no longer responsive in the hospital that she have the ability to be taken home for the rest of her time. It was only then that the doctors were very opposed to us taking her from the hospital. Not caring that it was her request that she not suffer the were not willing to let us take her home. Before her death she wanted to die at home with dignity. She didn’t want to be in the hospital. She truly believed that it would have been harder on the family to take care of her and she her like that she wanted to stay beautiful and not have her loved ones remember her as a â€Å"vegitable† laying in a hospital bed. This can be a really slippery slope. Who decides for the children or senile women? I truly The person requesting needs to  mentally stable and there needs to be safeguards against any family or doctors so as not to persuade the person in to euthanasia  projects in her article Let’s make a distinchin are qualifier. Terminal and suffering ,incurable, if theres a huge financial burden. Depression and mental

What Do Expect in English 101 Essay Example for Free

What Do Expect in English 101 Essay What are my expectations from this course? â€Å"Either write something worth reading or do something worth writing.† Benjamin Franklin. Reading is essential for everyday living, regardless of my insufficient skills. At the University of the Virgin Islands, English 101 class I’m determine to enhance my love for reading and build a stronger vocabulary foundation. I’ve never had a passion for reading. I was never surrounded by peers or family that influenced to me read. I always preferred to watch a movie filled with lies than read a book that would greatly impact my life. Now realizing how important reading is I’m stuck in the back playing catch up, which I highly dislike, especially as a freshman student. In English 101 I’m prepared to force myself to read, magazines, newspapers, novel and especially poems. I’ll love to understand when other say â€Å"I love reading because, allows me to go places Ive never been, experience new things, explore possibilities and learn something new.† I’m into fiction books I like knowing I can fly on the back of a dragon, turn my boyfriend into a toad, marry a prince, save a kingdom, cast a spell, swing a sword and talk to the gods. Just crazy things that never actually happens in reality. Loving reading makes everything possible, dreams come true, your imagination takes flight and your problems fade as you take on the problems of those you are reading about. To create a stabilize vocabulary foundation not only would I wait for my professor to assign work, I’ll try my best to take the responsibility of doing my own outside of class work. Gaining knowledge about words I’ll read words from the dictionary just like Malcolm X did. Previous I emphasized on my love for poetry never have I been capable to share my love because of my lack of words, but I’m already to make difference. Even though others may see it as a â€Å"dead and done† language, learning a bit of Latin would never hurt and I intend o n learning a bit of it. Also in this class I plan to familiarize myself with lots of familiarize magazines, books and puzzles to build my vocabulary. In conclusion, enhancing my passion for reading and building my vocabulary are my expectations in this class and my endeavor is to come climb to the top, I know my professor cannot do it alone, I have applied myself and I will succeed.

Toxicity and Autoactivation of Baits Experiment

Toxicity and Autoactivation of Baits Experiment Abstract Alternate splicing in exon 47 of the Purkinje cell calcium channel generates a splice variant with a five base pair insert (ggcag) before the stop codon in rat. This five base pair change the open reading frame of the exon 47 for resulting in an extended C-Terminal. Novel protein interaction at this region was hypothesised. Yeast Two Hybrid System was employed to screen against cDNA library to check for any protein interaction with 5 base pair insert region of exon 47. This project aimed to test the toxicity/ autoactivation of the baits in the yeast and to find the minimum concentration of 3-AT (3-amino-s-triole) at which it inhibits the HIS3 gene. The experimental result shows that there was no leaky expression of the HIS3 gene. The autoactivation/toxicity test results showed that the baits are less toxic than the control bait. The growth of non-interacting colonies in the Triple Drop Out media revealed that a more defined media should be used, demanding the repetition of experiment to obtain more convincing results. 1. Introduction 1.1. Nervous System The human nervous system consists of the Peripheral Nervous System (PNS) and the Central Nervous System (CNS). The PNS is formed of the cranial nerves and the spinal nerves. The central nervous system consists of the brain and the spinal cord. The brain can be divided into three major parts cerebrum, cerebellum and the brain stem. The cerebrum is divided into frontal lobe, parietal lobe, occipital lobe and the temporal lobe. The main function of cerebrum includes controlling of sensory organ, motor function, consciousness and imagining. The cerebellum is a uniform structure and its function is essential in movement and co- ordination of organs. The brain stem is made up of the mid brain, the pons and the medulla. The main functions of brain stem are transmission of information to and from the brain (Bear et al, 2001; Purves et al, 2004 and Thompson,1993). 1.2. Cells of CNS The brain consist mainly two types of cells nerve cells or neuron cells and the glial cells. The neuron are involved in the transport of electrical signals from the brain whereas the glial cells are thought to be the supporting cells of neurons by the uptake excess of neurotransmitter that are essential for signalling between neurons (Henn et al, 1971 and Purves et al, 2004) and plays a role in synaptogenesis of the neuron (Bacci et al, 1999). The glial cells are of three types: astrocytes, oligodentrocytes and the microglial cells. 1.2.1. Glial Cells Astrocytes are star shaped cells. The spatial arrangement of these cells between the capillaries and the neurons enables it in the modification of cellular responses, synaptic plasticity and survival of neurons (Abe et al, 2006 and Chen et al, 2003). Astrocytes important in glutamate transport, removal of free radical, controlling of haemostasis of brain and in maintaining a preferable environment for the active functioning of neurons by buffering K+ ions in their extracellular space (Chen et al, 2003; Gee et al, 2004 and Longuemare et al, 1999). Oligodentrocytes are type of glial cells that insulate the neuron with myelin sheath (Bear et al, 2001 and Lubetzki et al, 1993. The myelin sheath is a membrane which is made up of lipid and two proteins the proteolipoprotein (PLP) and the myelin basic protein (MBP). (Colman et al, 1982 and Boison et al, 1995). At regular intervals myelin sheath becomes thinner and is known as Nodes of Ranvier (Peter et al, 1966). These regions are the site for voltage gated sodium channels and a number of proteins. Microglial cells are the macrophages of the brain, which are formed in the bone marrow and are then transported to the brain by specialized protein called chemokines (Khoury et al, 2008) The study of chemokine receptors is one of the important research areas in the pathogenesis of Human Immuno Deficiency Virus. HIV can target microglial cells for their replication (Albright et al, 1999; Ghorpade et al, 1997 and Meer et al, 2000). Microglial cells are also studied for their inflammatory re sponses in the brain. The identification of role and mechanism by which microglial cells cause inflammation has paved path for finding targets and therapeutics for many diseases.(Bhatia, 2008; Huang et al 2008; Hwang et al, 2008 and Kim et al, 2008). 1.2.2. Neurons Neuron or the nerve cells are units of the nervous system involved in transfer of electrical signal between each other and to the effector cells. There are many types of nerve cells. Purkinje cells are one among them (Brown, 1991). The study of calcium ion channel of Purkinje cell is the subject of this project. The basic parts of neuron consist of a soma or cell body, axon, dendrites and neurites. All neurons are covered by the neuronal membrane. The soma or the cell body is similar to any other type of cell in the body. The axon is fibre that transport signal from the cell body to other neuron or to the target cell. The axons are covered by myelin sheath of the glial cells. The axon may be branched or unbranched. The main function of axon is to transfer the electrical signal from the axon hillock of soma throughout the axon known as the action potential and to transfer the signals to other cell in the form of chemical signal, the neurotransmitter (Purves et al, 2004 and Bear et al, 2001). The region of contact with other cells where release of neurotransmitter takes place is known as the synapse. The release of neurotransmitter is facilitated by synaptic vesicles of the presynaptic terminal (one which release chemical signal). The neurotransmitters are released by the synaptic vesicle in the space between pre synaptic and post synaptic terminal known as the synaptic cleft (Pu rves et al, 2004 and Brown et al, 1991). The neurotransmitters are then received by specific receptors of the post synaptic terminal which would generate an action potential in the cell. Apart from these receptors the ion channels of the cell membrane of the synaptic terminal also respond in the transfer of signal. Dendrites are branched fibres that arise from the cell. Their surface is lined with number of receptor to receive signals for the neuron (Brown,1991., Purves et al, 2004., Thompson,1993 and Bear et al, 2001). Purkinje cells are one of the largest types of neurons on the brain. They are found in the cerebellar region of the brain. The study of calcium ion channel of Purkinje cell is the subject of this project. Purkinje cells have a number of branches dendrites that receive synaptic inputs. As the dendrites receive signals it initiates a Ca2+ signal, which are important secondary messenger in the cells. The dendrites are the region for a calcium ion entry through the calcium ion channel. Similarly the soma contains K+ and Na+ channels(Schutter et al, 1994). These ions are of particular importance as their charge variation inside and outside the membrane trigger signalling in the cell. The transport of these ions is highly selective and they are maintained by the ion channel proteins of the Purkinje cell membrane and other neuronal membrane. These proteins form a pore for the transport of ions. Techniques such as the Patch clamp method have made the study of these ion channels easier (Bear et al, 2001). 1.3. Ion channel Ion channels are glycoprotein complex that allow specific ions through them. The proteins of ion channel are coded by different gene. More than 100 genes are known to code ion channels. The transportation of ion is important in generating action potential in the cell and is also important as the ions are second messengers in signalling. Diseases associated with the ion channel are known as channelopathies. Ion channels can be three major types voltage gated ion channel. Ligand gated ion channel and the stretch and heat activated ion channel (Purves et al.,2004). Voltage gated ion channels open and close on response to electrical potential. The voltage gated channels are made up of different protein sub unit. The subunits can move to open or close the channel (Horn, 2002). Depending on the type of ions they conduct they are further divided into Calcium channel, sodium channel and potassium channel. Ligand gated channels are those that respond to chemical signals. The ligand gated receptors are of five types nicotinic acetylcholine receptor (AChR), glutamate receptor, ÃŽ ³-aminobutyric acid (GABA), glycine-activated Channels and the ryanodine receptor(Stroud et al, 1990). Each of these receptors bind to specific ion and are found in different organs. The stretch and heat activated ion channel respond to heat or structural deformation of membrane (Purves et al, 2004). 1.4. Voltage Gated Calcium Channel (VGCC) Ca2+ ions are important secondary messenger in cells and play important role in biochemical pathways of cell. The level and entry of these Ca2+ ions in the cell is highly regulated. The regulations of these ions are controlled by the Voltage Gated Calcium Channel (Gribkoff et al, 2006). These VGCC are mainly found in excitatory cells such as the muscle cells and neurons. They exert their function by controlling muscle contraction, neurotransmitter release, neuronal plasticity, synapses, and neuronal excitability (Pietrobon, 2005 and Yang et al, 2005) . VGCC respond to membrane depolarization facilitating Ca2+ entry into the cell and thereby activating the signalling cascade of the cell (Yang et al, 2005). The normal functioning of the calcium channel protein is very important in a cell. Mutation in the gene coding channel protein, have been known to cause a number of diseases which include Timothy syndrome, Familial hemiplegic migraine type 2, episodic ataxia type 2, spinocerebellar ataxia type 6 and autism spectrum disorder which are grouped under â€Å"calcium channelopathies† (Bidaud et al, 2006 and Jen et al, 1999). Calcium channels also play a key role to mediate neuronal pain pathways (Gribkoff et al, 2006). A number of drugs have been known to block calcium channel and they are categorised as Calcium Channel Blockers. Verapamil was the first drug found to block Calcium Channel and later dihydropyridines (DHPs) class of drug was discovered to act as calcium channel blocker (Dolphin, 2006). DHPs are of much importance in studying the channel properties of the Dihydropyridine sensitive calcium channel. These DHP sensitive channels have dihydropyridine receptor for their bin ding (Campbell et al, 1988). Calcium Channel Blockers are now being found effective in the treatment of pain and hypertension (Atanassoff et al, 2000; Kize et al, 2001, and Thompson et al, 2001) but the question of safety in Coronary Heart Disease and the increased risk of cancer in patients remains unanswered (Eisenberg et al, 2004 and Fitzpatrick et al, 1997). 1.5. Calcium channel structure A calcium channel consists of five important subunits ÃŽ ±1. ÃŽ ±2, ÃŽ ², ÃŽ ´ and ÃŽ ³. The ÃŽ ±1 subunit is known as the pore forming complex (Yang et al, 2006). The ÃŽ ±1 subunit is a single polypeptide and its functions mainly include voltage sensing, gating and selective permeation (Horn et al, 2000). The structure of ÃŽ ±1 subunits consist of 24 segments (S1-S6) which constitute 4 domains, a C- terminal, N-terminal and Interlinkers. The linkers connecting domains are known as Loops and they are referred as loop I-II, loop II-III and loop III-IV depending on the domains they link (Dolphin, 2006). The intracellular loop of the ÃŽ ±1 subunit has interaction site for the binding ÃŽ ² subunit. The interaction can modulate the G- protein, an important second messenger in the cell (Dolphin, 1998). The specific binding of ÃŽ ² subunit to the tryptophan residue is important for controlling the gating of ÃŽ ±1 subunit of certain type of channels (Berrou, 2002). S4 is another important segment of the calcium channel. It is the voltage sensitive region of the calcium channel. S4 segment moves outward causing the channel to open by getting depolarised. S4 segment is positively charged due to the presence of arginine aminoacid making it voltage sensitive by translocation of the charges across the membrane (Sigworthl, 2003 and Horn et al, 2000). The S5, S6 and the linker connecting the S5 and S6 segment forms the boundaries ion conducting pore of the ÃŽ ±1 subunit. The ion conductance partly depends on the rotational movement of the S4 segment which either cause the S6 segment to open or c lose the pore (Horn et al, 2000). The ÃŽ ² subunits of the calcium channel are thought to be tissue specific and organ specific. Primarily they are of 4 different types, ÃŽ ²1, ÃŽ ²2, ÃŽ ²3 and ÃŽ ²4. Different isoforms of the ÃŽ ² subunits also do exist which include (CaB2a, CaB2b and CaB3) (Hullin et al, 1992 and Petegem et al, 2006). Their association with ÃŽ ± subunit is essential for modulation of VDI, CDI and CDF (Petegem et al, 2006). The ÃŽ ±2 subunit is also known as the ÃŽ ±2/ÃŽ ´ subunit as both the subunits are product of a single gene (Petegem et al, 2006). The ÃŽ ±2 and ÃŽ ´ subunits are linked together by disulphide bonds. Like other subunits ÃŽ ±2/ÃŽ ´ also exists as isoforms (Wang et al, 1999). They are known to play an important role in plasticity of neuron after a nerve injury and neuropathic pain processing (Luo et al, 2001). Gabapentin is a drug known to act on ÃŽ ±2/ÃŽ ´ subunit, but their binding affinity varies with different isoforms of the ÃŽ ´ subunit (Luo et al, 2001 and Luo et al, 2002).T he ÃŽ ³ subunit is found only in skeletal muscles. Their functional roles are yet to be discovered (Petegem et al, 2006). The C-terminus of calcium channel is a site for a number of protein- protein interactions in some channels. The expansion of the polyglutamine tract of the calcium channel is a major reason for the pathogenesis of the disease, Spino Cerebellar Ataxia 6 (SCA6). The cell death in SCA6 is thought to be caused by the poisoning of the nucleus by the localisation of C-terminal fragments (Kordasiewicz, 2006). 1.6. Calcium Channel Types Calcium channels account for the major amount of the calcium entry into the cell. The channel properties are tightly regulated to maintain Ca2+ concentration of the cell. The regulation was done through three well known processes. Voltage Dependent Inactivation (VDI) responsible for preventing entry of calcium into the cell. Calcium Dependent Inactivation (CD1) responsible for preventing entry of calcium into the cell whereas Calcium Dependent Facilitation (CDF) allows for the entry of calcium for signalling (Petegem et al, 2006). Based on the amount of current required to activate the channel the VDCC were termed either LVA channel (Low Voltage Activated) or HVA channel (High Voltage Activated). Later on due to the discovery of different current types, location of channel and sensitiveness to different types VDCC were broadly classified. Thus now 6 different types VDCC are known, in T type the current is transient, located in T-tubules and sensitive to dihydropyridine (DHP) (Dolphin, 2006). In L-Type the current is long lasting, found in neuron, heart and skeletal muscles and are sensitive to DHP. The N-Type stands for Non L Type or Neuronal and they are sensitive to ω-conotoxin GVIA (Petegem et al, 2006). The current found in Purkinje cells of the cerebral cortex were P-Type, they were sensitive to ω -agatoxin IVA. The Q-Type current are found in granular cells, however scientist consider P-Type and Q-Type to be same and are now term as P/Q- Type. The difference between the P Type and Q-Type is thoug ht to depend on the ÃŽ ² subunit to which it is associated(Dolphin., 2006). Another type of Residual current was also discovered which to date is not sensitive to any of the known toxin, this current is known as R-Type (Dolphin, 2006 and Petegem et al, 2006). 1.7. Calcium Channel Gene The alpha sub unit of the calcium channel are coded by 10 genes, therefore 10 different ÃŽ ±1 sub units are known. Of the ten types Cav 1.1 1.4 which is found in L-type, Cav 2.1 or the CavÃŽ ±1A is found in P/Q type channel, Cav2.2 is found in N type and Cav2.3 in R type channel. The Cav 3.1- 3.3 is found in T type channel. All these alpha subunit have one or more isoforms that would contribute to their functional diversity (Dolphin, 2006). The gene coding for the Cav 2.1, CACNA1A is found on the chromosome 19p13. This gene belongs to CACN family of gene that code for calcium channel. The gene characterised by the extension of CAG trinucleotide repeats. In humans the extension of the may vary from 4 to 18. Mutation of this gene cause diseases cause three major diseases FHM1 (Familial Hemiplegic Migraine 1), EA2 (Episodic Ataxia 2) and SCA6 (Spino Cerebellar Ataxia 6). Familial Hemiplegic Migraine is an autosomal dominant type of migraine caused by the missense mutation in CACNA1A. Three different mutations of CACNA1A cause FHM1 (Ducros et al, 1999). FHM1 affects the channel inactivation and the kinetics of the calcium channel (Kraus et al, 1997). The replacement of threonine with methionine is the mutation associated with FHM1. This mutation changes the channel structure causing more flow of calcium into cell. This ultimately results in the release of excess neurotransmitter (Ophoff et al, 1998). Episodic Ataxia 2 (EA2) is neurological disorder affecting the cerebellum and causing ataxia. The drug acetozolamide is known to be effective on EA2 (Ophoff et al, 1998). This disease has been found to have small but stable trinucleotide expansion but the role of the expansion is unknown for this disease (Jodice et al, 1997). The mutation in EA2 causes truncation of ÃŽ ±1A subunit which might cause a complete loss of the function of the channel (Wappl et al, 2002). 1.8. Spino Cerebellar Ataxia 6 Spino Cerebellar Ataxia 6 is also a neurodegenerative disease caused by the increase in number of CAG repeats in the CACNA1A gene (Tanaka et al, 2000). The number of trinucleotide repeat is between 22 and 28 in SCA6 (Riess, 1997). But it is not only the CAG repeats that are causing the disease. The ÃŽ ±1A have 6 isoforms and not all the isoforms are with the polyglutamine repeat. Therefore whether SCA6 is a channelopathy or Polyglutamine Disease remains a question among scientist (Frontali, 2006). The isoforms responsible for SCA6 is mainly limited to the C-Terminal. As the C-terminal is site for protein- protein interaction, changes in strength of interaction or changes in interacting partners tremendously affect the channel kinetics and other functional modification. As polyglutamine disease it cause toxic effect considered through aggregate formation (Pril et al, 2004). Comparison of number of repeats with other polyglutamine diseases where the repeat number is much high, the aggr egate formation alone cannot account for pathogenesis (Matsuyama et al, 1999). As a channelopathy the degeneration of Purkinje cell is caused by the poisoning of nucleus with the localised fragments of C-Terminal. The cleaved C terminal product is considered to have involved in signalling mechanism of the cell (Kordasiewcz et al, 2006). The isoforms of the C-Terminal of calcium channel are of considerable importance as the variation are found to be species specific (Kanumilli et al, 2005) and a few of them do not code for polyglutamine repeats. This invokes an interest in the C-terminal of the ÃŽ ±1A subunit of the calcium channel. The isoforms are formed by a process known as the pre-mRNA alternate splicing. 1.9. Splicing Transcription of messenger RNA (mRNA) from DNA and translation of proteins from mRNA forms the central dogma of molecular biology (Crick, 1970). These processes involves a series of important events, one among them is pre mRNA splicing. Before translation of protein, the mRNA needs to be processed by removing of non-coding introns. A human gene on an average consists of 8 introns. Splicing can lead to more than one type of mRNA from a single gene and consequently different protein isoforms (Faustino et al, 2003). Many different proteins are involved in splicing most importantly the spliceosome, a complex formed of small nuclear RNA (snRNA) and small nuclear ribonucleoproteins (Hagiwara et al, 2005 and Jurica et al, 2003). Small nuclear RNA can be of 5 important types U1, U2, U4, U5 and U6. All these in different combination target specific pre mRNA. The targeting is based on a number of factors which include phosphorylation of snRNAs, catalytic metal ions, enhancers, transcriptional coregulators and serine/arginine rich SR protein (Shi et al, 2006; Saba et al 2005; Auboeuf et al, 2007; Jurica et al, 2003; Hicks et al, 2005 and Manley et al, 2006). In general a spliceable introns has three regions splice donor, splice acceptor and a branch site. Most of the splice donor regions consist of AU nucleotide and the splice acceptor region consist of AG (Kenneth., 2005). Spliceosomes attach to these ends and by transesterification remove the introns, followed by the ligation of the exon (Rio,1993). Several mRNA have inherent splicing mechanism that does not require any spliceosome as they can splice themselves known as self splicing (Herrin et al, 1990 and Landthaler et al, 1999). Though most of the splicing is limited within the same mRNA, splicing also occurs between two different mRNAs by trans-splicing mechanism. The two mRNA exons called the mini exons were transcribed in different gene and were then combined to translate for a single protein (Bonen, 1993 and Bonen, 2008). Alternative splicing is a mechanism by which a few genes produce innumerable proteins that are diversified in structure and function. Nearly 75% of the human genes are involved in alternate splicing to give different protein isoforms (Hagiwara et al, 2005 and Stamm et al, 2004). The needs to understand alternate splicing have arised in almost all fields of biology. In evolutionary terms alternate splicing has a major role in the functional development of species right from the times of â€Å"RNA world†. The importance of isoforms has been understood through a number of studies. The Active and inactive forms of Sex lethal protein isoform are the determinants of sex of Drosophila (Herbert et al, 1999; Irimia et al, 2007 and Poole et al, 1998). Many different isoforms of normal proteins are discovered in cancer cells. These studies of these isoforms and their role have revealed some important diagnostic approach and cancer cell biomarkers (Brinkman, 2004; Skotheim et al, 2007 and Pampalakis et al, 2008). In the drug discovery process it is necessary to consider the mechanism of protein isoforms and pre mRNA splicing pathways and signalling molecules to identify new targets for drugs (Levanon et al, 2003 and Hagiwara et al, 2005). Alternative splicing in ion channels alter the conductance and functional properties of the channel. Splicing has been known in voltage gated sodium channel, voltage gated calcium channel, ligand gated ion channel and in calcium gated potassium channel. Although the ion channels differ in their properties, all share some basic function. These ion channels have multiple splicing site through which their channelling properties are regulated based on the organs where these channels are located (Copley, 2004; Raymond et al, 2004; Sarao et al, 1991 and Schaller et al, 1992). 1.10. CaV2.1 splice variants Variants in calcium channel protein, in particular the 47 exon of the c-terminal is the basis of this study. Splicing in calcium channel occurs at distinct region such as the loops between the II-III domains which is the major interacting site for ryodine receptor. Two isoforms BI and rbA are found in loop II-III of rat and rabbit. They differ in their interacting ability towards syntaxin and synaptotagmin proteins. These proteins can modulate the Ca+ influx of the neuron (Charvin et al, 1997 and Rettig et al, 1996). Site specific variations are found in exons 9, 31, 44, 46 and the extreme C terminus e47 (shown in Fig 3)(Kanumilli et al, 2005). The C- termini of calcium channels are involved in the modulation of G-proteins, molecular switching of calmodulin and are the site for protein-protein interaction. So a single amino acid change can potentially change the gating property and other function of the channel and its interacting partners (Chaudhuri et al, 2004; Gray et al, 2007; Krovetz et al, 2000 and Ligon et al, 1997) splice variants were known to occur in the C- terminal the calcium channel. A 5 base pair insertion (ggcag) was reported in pancreatic islets of rats a variant already known in human (Ligon et al, 1997). This 5 base pair insertion is expected to alter the length on the c terminal and hence channel property as it found before the stop codon, which means a change in the reading frame. The existence of variants with and without the 5 base pair (ggcag) insert before the stop codon of rat Purkinje cell is confirmed by Kanumilli et al (2005). Another independent study with mouse by Tsunemi et al (2001) also confirmed the 5 base pair insert. In addition, variants without the stop codon and a ggcag insert, 150 nucleotide deletions in the 5- end of the C- terminal is reported in mouse (Kanumilli et al, 2005). The absence of stop codon was also observed in the study by Tsunemi et al (2001) in mouse. Richards et al (2007) obtained similar results with rat Purkinje cell, the sequence of exon 47 were same as the rat pancreatic cells except for variations in other exons. However variation in the number of amino acid (156 residues, 153 residues and 115 residues) coded by exon 47 were observed in different clones. The 156 amino acid length was also reported by Ligon et al (1998). These finding and most other results describe the calcium channel properties in terms of activation or inactivation kinetics. However no protein- protein interaction study is available till date for the exon 47 with five base pair (ggcag) inclusion before the stop codon. The need for studies at the protein-protein interaction level is necessary which is evident from the studies of Dolphin(2006), Richards et al (2007), Sandoz et al (2001) and Kanumilli et al (2005). This study was aimed at studying possible protein-protein interaction for exon 47 of rat Purkinje cell. Then linking the interacting the protein to already known biochemical pathway is expected to give more insight the channel and possibly a new perspective in the treatment of SCA6. 1.11. Protein protein interaction studies Protein-Protein interaction is an important part in all biological process. A protein- protein interaction can altogether change the binding characteristics, kinetic property and their catalytic ability (Eisenberg et al, 2000). A number of methods have been developed and used to study protein-protein interaction. These methods can be the detection and analysis of interaction or can be screening against a family of proteins. Detection methods are mostly used to confirm and study known interaction. These methods include Protein Affinity chromatography, Affinity Blotting, Coimmunoprecipitation and Cross- linking. The screening methods include protein probing, phage display and the Yeast Two Hybrid System (Y2H) (Phizicky et al, 1995). Bioinformatics tools such as protein docking are also important in predicting the protein interactions (Smith et al, 2002). 1.12. Yeast Two Hybrid System (Y2H) Yeast two hybrid system is the most widely used protein screening methods. The requirement of an interaction between two domains DNA Binding Domain (DNA-BD) and Activation Domain (AD) for the expression of a reporter gene (lac-z) in yeast is being exploited in Y2H. The lac-z gene expression gives our ÃŽ ²-galactosidase enzyme which can be observed by colour change confirming interaction (as shown in Fig 4) (Criekinge et al, 1999). The protein of interest (bait) is usually fused with the BD and the interacting protein or the library protein is fused with activation domain. The protein of interest is normally termed as bait and the interacting protein is called a prey. Bacterial plasmid can be easily constructed to express fusion protein of interest. The bacterial shuttle vector can be isolated and transfected into the yeast for their expression. On expression the DNA-BD fusion protein will bind to the upstream activation sequence of the reporter gene. Two types of Y2H are known one is the GAL4 based system and the other is the Lex A based system. In Lex yeast two hybrid system the prey is fused with the Lex A binding domain. The specifically interacts with the Lex A operator upstream sequence which is the part of the promoter for reporter gene. The prey will be fused with the GAL 4 protein. In the GAL 4 system instead of Lex A the GAL 4 promoter will be used. Both the systems have their advantages and their dis advantages (Criekinge et al, 1999 and Luban et al, 1995) The yeast strain L40 is compatible with LexA operator and the GAL 4 promoter system. Most Y2H methods are done more than one reporter gene for more selectivity. HIS3 gene is one such reporter that is used for the nutritional selection of the cells. HIS3 reporter expression needs the interaction of proteins. So cells would not grow in a media lacking histidine if no interactions take place. Similar nutritional selections are also used in cell containing only the baits or only the prey. The nutritional selection for bait is tryptophan and for the prey is leucine. It is therefore important to use a defined media. A positive interaction between bait and the prey will allow growth in the Triple Drop Out media (TDO/ -His/-Leu/-Trp) (Criekinge et al, 1999 and Luban et al, 1995) The use of histidine reporter gene can sometimes account for leaky expression. In which case 3-AT (3-amino-s-triole) a competitive inhibitor of histidine can be tried in various concentration to find a minimum concentration at which cells grow and the enzyme is inhibited. Cells growing concentration of 100mM concentration cannot be used as baits (Criekinge et al, 1999). Toxicity caused by bait can inhibit the growth of yeast (Zhong et al, 2003). Toxicity tests have to be carried out to after the baits are designed. Autoactivation of the baits should be checked before proceeding to the, library screening as nearly 5% of the protein can initiate transcription without an interactor (Criekinge et al, 1999). After the library screening the plasmids can be isolated and used to transform bacterial cells. The interaction also has to be confirmed and isolated by techniques such as coimmunoprecipitation. 2. Aim This study was undertaken as a part of the project by Dr. Claire Palmer in finding novel protein-protein interaction for 5-base pair insert in exon 47 of rat cerebellar Purkinje cell(AF051526). Yeast 2 hybrid system was employed to study interaction. Accordingly two protein baits 5inSER and NLSER were constructed by colleague Surya to screen against library protein. Baits 5inSER is a 472 base pair length protein with ggcag NLSER is a 397 base pair length protein without the Nuclear Localisation Sequence. It was constructed to find the significance of the nuclear localisation signal (Surya, 2008). The aims of the project are To test for toxicity and autoactivation of baits. To determine the concentration of 3-AT at which the expression of Histidine gene is inhibited. Control mating experiment. 3. Materials and Methods 3.1. Control Mating 3.1.1. Control strains The control mating experiments were done prior to the library screen. The positive control yeast strains AH109 with the bait [pGBKT7-53] and Y187 with the target [pGADT7-T] , glycerol stock were provided. For negative control the bait strain was L40 with bait pBTM116/GluR2 and the target was the same Y187[pGADT7-T] The negative control bait was obtained by the transformation of L40 with the plasmids isolated from provided E.Coli cultures. 3.1.2. Small Scale Yeast Transformation A colony of Saccharomyces cerevisiae L40 yeast was inoculated into 10ml of YPAD media. It was left overnight in a shaking incubator (200rpm) at 30à ¢Ã‚ Ã‚ ° C. The overnight culture was diluted in 50 ml of YPAD to an OD600 Toxicity and Autoactivation of Baits Experiment Toxicity and Autoactivation of Baits Experiment Abstract Alternate splicing in exon 47 of the Purkinje cell calcium channel generates a splice variant with a five base pair insert (ggcag) before the stop codon in rat. This five base pair change the open reading frame of the exon 47 for resulting in an extended C-Terminal. Novel protein interaction at this region was hypothesised. Yeast Two Hybrid System was employed to screen against cDNA library to check for any protein interaction with 5 base pair insert region of exon 47. This project aimed to test the toxicity/ autoactivation of the baits in the yeast and to find the minimum concentration of 3-AT (3-amino-s-triole) at which it inhibits the HIS3 gene. The experimental result shows that there was no leaky expression of the HIS3 gene. The autoactivation/toxicity test results showed that the baits are less toxic than the control bait. The growth of non-interacting colonies in the Triple Drop Out media revealed that a more defined media should be used, demanding the repetition of experiment to obtain more convincing results. 1. Introduction 1.1. Nervous System The human nervous system consists of the Peripheral Nervous System (PNS) and the Central Nervous System (CNS). The PNS is formed of the cranial nerves and the spinal nerves. The central nervous system consists of the brain and the spinal cord. The brain can be divided into three major parts cerebrum, cerebellum and the brain stem. The cerebrum is divided into frontal lobe, parietal lobe, occipital lobe and the temporal lobe. The main function of cerebrum includes controlling of sensory organ, motor function, consciousness and imagining. The cerebellum is a uniform structure and its function is essential in movement and co- ordination of organs. The brain stem is made up of the mid brain, the pons and the medulla. The main functions of brain stem are transmission of information to and from the brain (Bear et al, 2001; Purves et al, 2004 and Thompson,1993). 1.2. Cells of CNS The brain consist mainly two types of cells nerve cells or neuron cells and the glial cells. The neuron are involved in the transport of electrical signals from the brain whereas the glial cells are thought to be the supporting cells of neurons by the uptake excess of neurotransmitter that are essential for signalling between neurons (Henn et al, 1971 and Purves et al, 2004) and plays a role in synaptogenesis of the neuron (Bacci et al, 1999). The glial cells are of three types: astrocytes, oligodentrocytes and the microglial cells. 1.2.1. Glial Cells Astrocytes are star shaped cells. The spatial arrangement of these cells between the capillaries and the neurons enables it in the modification of cellular responses, synaptic plasticity and survival of neurons (Abe et al, 2006 and Chen et al, 2003). Astrocytes important in glutamate transport, removal of free radical, controlling of haemostasis of brain and in maintaining a preferable environment for the active functioning of neurons by buffering K+ ions in their extracellular space (Chen et al, 2003; Gee et al, 2004 and Longuemare et al, 1999). Oligodentrocytes are type of glial cells that insulate the neuron with myelin sheath (Bear et al, 2001 and Lubetzki et al, 1993. The myelin sheath is a membrane which is made up of lipid and two proteins the proteolipoprotein (PLP) and the myelin basic protein (MBP). (Colman et al, 1982 and Boison et al, 1995). At regular intervals myelin sheath becomes thinner and is known as Nodes of Ranvier (Peter et al, 1966). These regions are the site for voltage gated sodium channels and a number of proteins. Microglial cells are the macrophages of the brain, which are formed in the bone marrow and are then transported to the brain by specialized protein called chemokines (Khoury et al, 2008) The study of chemokine receptors is one of the important research areas in the pathogenesis of Human Immuno Deficiency Virus. HIV can target microglial cells for their replication (Albright et al, 1999; Ghorpade et al, 1997 and Meer et al, 2000). Microglial cells are also studied for their inflammatory re sponses in the brain. The identification of role and mechanism by which microglial cells cause inflammation has paved path for finding targets and therapeutics for many diseases.(Bhatia, 2008; Huang et al 2008; Hwang et al, 2008 and Kim et al, 2008). 1.2.2. Neurons Neuron or the nerve cells are units of the nervous system involved in transfer of electrical signal between each other and to the effector cells. There are many types of nerve cells. Purkinje cells are one among them (Brown, 1991). The study of calcium ion channel of Purkinje cell is the subject of this project. The basic parts of neuron consist of a soma or cell body, axon, dendrites and neurites. All neurons are covered by the neuronal membrane. The soma or the cell body is similar to any other type of cell in the body. The axon is fibre that transport signal from the cell body to other neuron or to the target cell. The axons are covered by myelin sheath of the glial cells. The axon may be branched or unbranched. The main function of axon is to transfer the electrical signal from the axon hillock of soma throughout the axon known as the action potential and to transfer the signals to other cell in the form of chemical signal, the neurotransmitter (Purves et al, 2004 and Bear et al, 2001). The region of contact with other cells where release of neurotransmitter takes place is known as the synapse. The release of neurotransmitter is facilitated by synaptic vesicles of the presynaptic terminal (one which release chemical signal). The neurotransmitters are released by the synaptic vesicle in the space between pre synaptic and post synaptic terminal known as the synaptic cleft (Pu rves et al, 2004 and Brown et al, 1991). The neurotransmitters are then received by specific receptors of the post synaptic terminal which would generate an action potential in the cell. Apart from these receptors the ion channels of the cell membrane of the synaptic terminal also respond in the transfer of signal. Dendrites are branched fibres that arise from the cell. Their surface is lined with number of receptor to receive signals for the neuron (Brown,1991., Purves et al, 2004., Thompson,1993 and Bear et al, 2001). Purkinje cells are one of the largest types of neurons on the brain. They are found in the cerebellar region of the brain. The study of calcium ion channel of Purkinje cell is the subject of this project. Purkinje cells have a number of branches dendrites that receive synaptic inputs. As the dendrites receive signals it initiates a Ca2+ signal, which are important secondary messenger in the cells. The dendrites are the region for a calcium ion entry through the calcium ion channel. Similarly the soma contains K+ and Na+ channels(Schutter et al, 1994). These ions are of particular importance as their charge variation inside and outside the membrane trigger signalling in the cell. The transport of these ions is highly selective and they are maintained by the ion channel proteins of the Purkinje cell membrane and other neuronal membrane. These proteins form a pore for the transport of ions. Techniques such as the Patch clamp method have made the study of these ion channels easier (Bear et al, 2001). 1.3. Ion channel Ion channels are glycoprotein complex that allow specific ions through them. The proteins of ion channel are coded by different gene. More than 100 genes are known to code ion channels. The transportation of ion is important in generating action potential in the cell and is also important as the ions are second messengers in signalling. Diseases associated with the ion channel are known as channelopathies. Ion channels can be three major types voltage gated ion channel. Ligand gated ion channel and the stretch and heat activated ion channel (Purves et al.,2004). Voltage gated ion channels open and close on response to electrical potential. The voltage gated channels are made up of different protein sub unit. The subunits can move to open or close the channel (Horn, 2002). Depending on the type of ions they conduct they are further divided into Calcium channel, sodium channel and potassium channel. Ligand gated channels are those that respond to chemical signals. The ligand gated receptors are of five types nicotinic acetylcholine receptor (AChR), glutamate receptor, ÃŽ ³-aminobutyric acid (GABA), glycine-activated Channels and the ryanodine receptor(Stroud et al, 1990). Each of these receptors bind to specific ion and are found in different organs. The stretch and heat activated ion channel respond to heat or structural deformation of membrane (Purves et al, 2004). 1.4. Voltage Gated Calcium Channel (VGCC) Ca2+ ions are important secondary messenger in cells and play important role in biochemical pathways of cell. The level and entry of these Ca2+ ions in the cell is highly regulated. The regulations of these ions are controlled by the Voltage Gated Calcium Channel (Gribkoff et al, 2006). These VGCC are mainly found in excitatory cells such as the muscle cells and neurons. They exert their function by controlling muscle contraction, neurotransmitter release, neuronal plasticity, synapses, and neuronal excitability (Pietrobon, 2005 and Yang et al, 2005) . VGCC respond to membrane depolarization facilitating Ca2+ entry into the cell and thereby activating the signalling cascade of the cell (Yang et al, 2005). The normal functioning of the calcium channel protein is very important in a cell. Mutation in the gene coding channel protein, have been known to cause a number of diseases which include Timothy syndrome, Familial hemiplegic migraine type 2, episodic ataxia type 2, spinocerebellar ataxia type 6 and autism spectrum disorder which are grouped under â€Å"calcium channelopathies† (Bidaud et al, 2006 and Jen et al, 1999). Calcium channels also play a key role to mediate neuronal pain pathways (Gribkoff et al, 2006). A number of drugs have been known to block calcium channel and they are categorised as Calcium Channel Blockers. Verapamil was the first drug found to block Calcium Channel and later dihydropyridines (DHPs) class of drug was discovered to act as calcium channel blocker (Dolphin, 2006). DHPs are of much importance in studying the channel properties of the Dihydropyridine sensitive calcium channel. These DHP sensitive channels have dihydropyridine receptor for their bin ding (Campbell et al, 1988). Calcium Channel Blockers are now being found effective in the treatment of pain and hypertension (Atanassoff et al, 2000; Kize et al, 2001, and Thompson et al, 2001) but the question of safety in Coronary Heart Disease and the increased risk of cancer in patients remains unanswered (Eisenberg et al, 2004 and Fitzpatrick et al, 1997). 1.5. Calcium channel structure A calcium channel consists of five important subunits ÃŽ ±1. ÃŽ ±2, ÃŽ ², ÃŽ ´ and ÃŽ ³. The ÃŽ ±1 subunit is known as the pore forming complex (Yang et al, 2006). The ÃŽ ±1 subunit is a single polypeptide and its functions mainly include voltage sensing, gating and selective permeation (Horn et al, 2000). The structure of ÃŽ ±1 subunits consist of 24 segments (S1-S6) which constitute 4 domains, a C- terminal, N-terminal and Interlinkers. The linkers connecting domains are known as Loops and they are referred as loop I-II, loop II-III and loop III-IV depending on the domains they link (Dolphin, 2006). The intracellular loop of the ÃŽ ±1 subunit has interaction site for the binding ÃŽ ² subunit. The interaction can modulate the G- protein, an important second messenger in the cell (Dolphin, 1998). The specific binding of ÃŽ ² subunit to the tryptophan residue is important for controlling the gating of ÃŽ ±1 subunit of certain type of channels (Berrou, 2002). S4 is another important segment of the calcium channel. It is the voltage sensitive region of the calcium channel. S4 segment moves outward causing the channel to open by getting depolarised. S4 segment is positively charged due to the presence of arginine aminoacid making it voltage sensitive by translocation of the charges across the membrane (Sigworthl, 2003 and Horn et al, 2000). The S5, S6 and the linker connecting the S5 and S6 segment forms the boundaries ion conducting pore of the ÃŽ ±1 subunit. The ion conductance partly depends on the rotational movement of the S4 segment which either cause the S6 segment to open or c lose the pore (Horn et al, 2000). The ÃŽ ² subunits of the calcium channel are thought to be tissue specific and organ specific. Primarily they are of 4 different types, ÃŽ ²1, ÃŽ ²2, ÃŽ ²3 and ÃŽ ²4. Different isoforms of the ÃŽ ² subunits also do exist which include (CaB2a, CaB2b and CaB3) (Hullin et al, 1992 and Petegem et al, 2006). Their association with ÃŽ ± subunit is essential for modulation of VDI, CDI and CDF (Petegem et al, 2006). The ÃŽ ±2 subunit is also known as the ÃŽ ±2/ÃŽ ´ subunit as both the subunits are product of a single gene (Petegem et al, 2006). The ÃŽ ±2 and ÃŽ ´ subunits are linked together by disulphide bonds. Like other subunits ÃŽ ±2/ÃŽ ´ also exists as isoforms (Wang et al, 1999). They are known to play an important role in plasticity of neuron after a nerve injury and neuropathic pain processing (Luo et al, 2001). Gabapentin is a drug known to act on ÃŽ ±2/ÃŽ ´ subunit, but their binding affinity varies with different isoforms of the ÃŽ ´ subunit (Luo et al, 2001 and Luo et al, 2002).T he ÃŽ ³ subunit is found only in skeletal muscles. Their functional roles are yet to be discovered (Petegem et al, 2006). The C-terminus of calcium channel is a site for a number of protein- protein interactions in some channels. The expansion of the polyglutamine tract of the calcium channel is a major reason for the pathogenesis of the disease, Spino Cerebellar Ataxia 6 (SCA6). The cell death in SCA6 is thought to be caused by the poisoning of the nucleus by the localisation of C-terminal fragments (Kordasiewicz, 2006). 1.6. Calcium Channel Types Calcium channels account for the major amount of the calcium entry into the cell. The channel properties are tightly regulated to maintain Ca2+ concentration of the cell. The regulation was done through three well known processes. Voltage Dependent Inactivation (VDI) responsible for preventing entry of calcium into the cell. Calcium Dependent Inactivation (CD1) responsible for preventing entry of calcium into the cell whereas Calcium Dependent Facilitation (CDF) allows for the entry of calcium for signalling (Petegem et al, 2006). Based on the amount of current required to activate the channel the VDCC were termed either LVA channel (Low Voltage Activated) or HVA channel (High Voltage Activated). Later on due to the discovery of different current types, location of channel and sensitiveness to different types VDCC were broadly classified. Thus now 6 different types VDCC are known, in T type the current is transient, located in T-tubules and sensitive to dihydropyridine (DHP) (Dolphin, 2006). In L-Type the current is long lasting, found in neuron, heart and skeletal muscles and are sensitive to DHP. The N-Type stands for Non L Type or Neuronal and they are sensitive to ω-conotoxin GVIA (Petegem et al, 2006). The current found in Purkinje cells of the cerebral cortex were P-Type, they were sensitive to ω -agatoxin IVA. The Q-Type current are found in granular cells, however scientist consider P-Type and Q-Type to be same and are now term as P/Q- Type. The difference between the P Type and Q-Type is thoug ht to depend on the ÃŽ ² subunit to which it is associated(Dolphin., 2006). Another type of Residual current was also discovered which to date is not sensitive to any of the known toxin, this current is known as R-Type (Dolphin, 2006 and Petegem et al, 2006). 1.7. Calcium Channel Gene The alpha sub unit of the calcium channel are coded by 10 genes, therefore 10 different ÃŽ ±1 sub units are known. Of the ten types Cav 1.1 1.4 which is found in L-type, Cav 2.1 or the CavÃŽ ±1A is found in P/Q type channel, Cav2.2 is found in N type and Cav2.3 in R type channel. The Cav 3.1- 3.3 is found in T type channel. All these alpha subunit have one or more isoforms that would contribute to their functional diversity (Dolphin, 2006). The gene coding for the Cav 2.1, CACNA1A is found on the chromosome 19p13. This gene belongs to CACN family of gene that code for calcium channel. The gene characterised by the extension of CAG trinucleotide repeats. In humans the extension of the may vary from 4 to 18. Mutation of this gene cause diseases cause three major diseases FHM1 (Familial Hemiplegic Migraine 1), EA2 (Episodic Ataxia 2) and SCA6 (Spino Cerebellar Ataxia 6). Familial Hemiplegic Migraine is an autosomal dominant type of migraine caused by the missense mutation in CACNA1A. Three different mutations of CACNA1A cause FHM1 (Ducros et al, 1999). FHM1 affects the channel inactivation and the kinetics of the calcium channel (Kraus et al, 1997). The replacement of threonine with methionine is the mutation associated with FHM1. This mutation changes the channel structure causing more flow of calcium into cell. This ultimately results in the release of excess neurotransmitter (Ophoff et al, 1998). Episodic Ataxia 2 (EA2) is neurological disorder affecting the cerebellum and causing ataxia. The drug acetozolamide is known to be effective on EA2 (Ophoff et al, 1998). This disease has been found to have small but stable trinucleotide expansion but the role of the expansion is unknown for this disease (Jodice et al, 1997). The mutation in EA2 causes truncation of ÃŽ ±1A subunit which might cause a complete loss of the function of the channel (Wappl et al, 2002). 1.8. Spino Cerebellar Ataxia 6 Spino Cerebellar Ataxia 6 is also a neurodegenerative disease caused by the increase in number of CAG repeats in the CACNA1A gene (Tanaka et al, 2000). The number of trinucleotide repeat is between 22 and 28 in SCA6 (Riess, 1997). But it is not only the CAG repeats that are causing the disease. The ÃŽ ±1A have 6 isoforms and not all the isoforms are with the polyglutamine repeat. Therefore whether SCA6 is a channelopathy or Polyglutamine Disease remains a question among scientist (Frontali, 2006). The isoforms responsible for SCA6 is mainly limited to the C-Terminal. As the C-terminal is site for protein- protein interaction, changes in strength of interaction or changes in interacting partners tremendously affect the channel kinetics and other functional modification. As polyglutamine disease it cause toxic effect considered through aggregate formation (Pril et al, 2004). Comparison of number of repeats with other polyglutamine diseases where the repeat number is much high, the aggr egate formation alone cannot account for pathogenesis (Matsuyama et al, 1999). As a channelopathy the degeneration of Purkinje cell is caused by the poisoning of nucleus with the localised fragments of C-Terminal. The cleaved C terminal product is considered to have involved in signalling mechanism of the cell (Kordasiewcz et al, 2006). The isoforms of the C-Terminal of calcium channel are of considerable importance as the variation are found to be species specific (Kanumilli et al, 2005) and a few of them do not code for polyglutamine repeats. This invokes an interest in the C-terminal of the ÃŽ ±1A subunit of the calcium channel. The isoforms are formed by a process known as the pre-mRNA alternate splicing. 1.9. Splicing Transcription of messenger RNA (mRNA) from DNA and translation of proteins from mRNA forms the central dogma of molecular biology (Crick, 1970). These processes involves a series of important events, one among them is pre mRNA splicing. Before translation of protein, the mRNA needs to be processed by removing of non-coding introns. A human gene on an average consists of 8 introns. Splicing can lead to more than one type of mRNA from a single gene and consequently different protein isoforms (Faustino et al, 2003). Many different proteins are involved in splicing most importantly the spliceosome, a complex formed of small nuclear RNA (snRNA) and small nuclear ribonucleoproteins (Hagiwara et al, 2005 and Jurica et al, 2003). Small nuclear RNA can be of 5 important types U1, U2, U4, U5 and U6. All these in different combination target specific pre mRNA. The targeting is based on a number of factors which include phosphorylation of snRNAs, catalytic metal ions, enhancers, transcriptional coregulators and serine/arginine rich SR protein (Shi et al, 2006; Saba et al 2005; Auboeuf et al, 2007; Jurica et al, 2003; Hicks et al, 2005 and Manley et al, 2006). In general a spliceable introns has three regions splice donor, splice acceptor and a branch site. Most of the splice donor regions consist of AU nucleotide and the splice acceptor region consist of AG (Kenneth., 2005). Spliceosomes attach to these ends and by transesterification remove the introns, followed by the ligation of the exon (Rio,1993). Several mRNA have inherent splicing mechanism that does not require any spliceosome as they can splice themselves known as self splicing (Herrin et al, 1990 and Landthaler et al, 1999). Though most of the splicing is limited within the same mRNA, splicing also occurs between two different mRNAs by trans-splicing mechanism. The two mRNA exons called the mini exons were transcribed in different gene and were then combined to translate for a single protein (Bonen, 1993 and Bonen, 2008). Alternative splicing is a mechanism by which a few genes produce innumerable proteins that are diversified in structure and function. Nearly 75% of the human genes are involved in alternate splicing to give different protein isoforms (Hagiwara et al, 2005 and Stamm et al, 2004). The needs to understand alternate splicing have arised in almost all fields of biology. In evolutionary terms alternate splicing has a major role in the functional development of species right from the times of â€Å"RNA world†. The importance of isoforms has been understood through a number of studies. The Active and inactive forms of Sex lethal protein isoform are the determinants of sex of Drosophila (Herbert et al, 1999; Irimia et al, 2007 and Poole et al, 1998). Many different isoforms of normal proteins are discovered in cancer cells. These studies of these isoforms and their role have revealed some important diagnostic approach and cancer cell biomarkers (Brinkman, 2004; Skotheim et al, 2007 and Pampalakis et al, 2008). In the drug discovery process it is necessary to consider the mechanism of protein isoforms and pre mRNA splicing pathways and signalling molecules to identify new targets for drugs (Levanon et al, 2003 and Hagiwara et al, 2005). Alternative splicing in ion channels alter the conductance and functional properties of the channel. Splicing has been known in voltage gated sodium channel, voltage gated calcium channel, ligand gated ion channel and in calcium gated potassium channel. Although the ion channels differ in their properties, all share some basic function. These ion channels have multiple splicing site through which their channelling properties are regulated based on the organs where these channels are located (Copley, 2004; Raymond et al, 2004; Sarao et al, 1991 and Schaller et al, 1992). 1.10. CaV2.1 splice variants Variants in calcium channel protein, in particular the 47 exon of the c-terminal is the basis of this study. Splicing in calcium channel occurs at distinct region such as the loops between the II-III domains which is the major interacting site for ryodine receptor. Two isoforms BI and rbA are found in loop II-III of rat and rabbit. They differ in their interacting ability towards syntaxin and synaptotagmin proteins. These proteins can modulate the Ca+ influx of the neuron (Charvin et al, 1997 and Rettig et al, 1996). Site specific variations are found in exons 9, 31, 44, 46 and the extreme C terminus e47 (shown in Fig 3)(Kanumilli et al, 2005). The C- termini of calcium channels are involved in the modulation of G-proteins, molecular switching of calmodulin and are the site for protein-protein interaction. So a single amino acid change can potentially change the gating property and other function of the channel and its interacting partners (Chaudhuri et al, 2004; Gray et al, 2007; Krovetz et al, 2000 and Ligon et al, 1997) splice variants were known to occur in the C- terminal the calcium channel. A 5 base pair insertion (ggcag) was reported in pancreatic islets of rats a variant already known in human (Ligon et al, 1997). This 5 base pair insertion is expected to alter the length on the c terminal and hence channel property as it found before the stop codon, which means a change in the reading frame. The existence of variants with and without the 5 base pair (ggcag) insert before the stop codon of rat Purkinje cell is confirmed by Kanumilli et al (2005). Another independent study with mouse by Tsunemi et al (2001) also confirmed the 5 base pair insert. In addition, variants without the stop codon and a ggcag insert, 150 nucleotide deletions in the 5- end of the C- terminal is reported in mouse (Kanumilli et al, 2005). The absence of stop codon was also observed in the study by Tsunemi et al (2001) in mouse. Richards et al (2007) obtained similar results with rat Purkinje cell, the sequence of exon 47 were same as the rat pancreatic cells except for variations in other exons. However variation in the number of amino acid (156 residues, 153 residues and 115 residues) coded by exon 47 were observed in different clones. The 156 amino acid length was also reported by Ligon et al (1998). These finding and most other results describe the calcium channel properties in terms of activation or inactivation kinetics. However no protein- protein interaction study is available till date for the exon 47 with five base pair (ggcag) inclusion before the stop codon. The need for studies at the protein-protein interaction level is necessary which is evident from the studies of Dolphin(2006), Richards et al (2007), Sandoz et al (2001) and Kanumilli et al (2005). This study was aimed at studying possible protein-protein interaction for exon 47 of rat Purkinje cell. Then linking the interacting the protein to already known biochemical pathway is expected to give more insight the channel and possibly a new perspective in the treatment of SCA6. 1.11. Protein protein interaction studies Protein-Protein interaction is an important part in all biological process. A protein- protein interaction can altogether change the binding characteristics, kinetic property and their catalytic ability (Eisenberg et al, 2000). A number of methods have been developed and used to study protein-protein interaction. These methods can be the detection and analysis of interaction or can be screening against a family of proteins. Detection methods are mostly used to confirm and study known interaction. These methods include Protein Affinity chromatography, Affinity Blotting, Coimmunoprecipitation and Cross- linking. The screening methods include protein probing, phage display and the Yeast Two Hybrid System (Y2H) (Phizicky et al, 1995). Bioinformatics tools such as protein docking are also important in predicting the protein interactions (Smith et al, 2002). 1.12. Yeast Two Hybrid System (Y2H) Yeast two hybrid system is the most widely used protein screening methods. The requirement of an interaction between two domains DNA Binding Domain (DNA-BD) and Activation Domain (AD) for the expression of a reporter gene (lac-z) in yeast is being exploited in Y2H. The lac-z gene expression gives our ÃŽ ²-galactosidase enzyme which can be observed by colour change confirming interaction (as shown in Fig 4) (Criekinge et al, 1999). The protein of interest (bait) is usually fused with the BD and the interacting protein or the library protein is fused with activation domain. The protein of interest is normally termed as bait and the interacting protein is called a prey. Bacterial plasmid can be easily constructed to express fusion protein of interest. The bacterial shuttle vector can be isolated and transfected into the yeast for their expression. On expression the DNA-BD fusion protein will bind to the upstream activation sequence of the reporter gene. Two types of Y2H are known one is the GAL4 based system and the other is the Lex A based system. In Lex yeast two hybrid system the prey is fused with the Lex A binding domain. The specifically interacts with the Lex A operator upstream sequence which is the part of the promoter for reporter gene. The prey will be fused with the GAL 4 protein. In the GAL 4 system instead of Lex A the GAL 4 promoter will be used. Both the systems have their advantages and their dis advantages (Criekinge et al, 1999 and Luban et al, 1995) The yeast strain L40 is compatible with LexA operator and the GAL 4 promoter system. Most Y2H methods are done more than one reporter gene for more selectivity. HIS3 gene is one such reporter that is used for the nutritional selection of the cells. HIS3 reporter expression needs the interaction of proteins. So cells would not grow in a media lacking histidine if no interactions take place. Similar nutritional selections are also used in cell containing only the baits or only the prey. The nutritional selection for bait is tryptophan and for the prey is leucine. It is therefore important to use a defined media. A positive interaction between bait and the prey will allow growth in the Triple Drop Out media (TDO/ -His/-Leu/-Trp) (Criekinge et al, 1999 and Luban et al, 1995) The use of histidine reporter gene can sometimes account for leaky expression. In which case 3-AT (3-amino-s-triole) a competitive inhibitor of histidine can be tried in various concentration to find a minimum concentration at which cells grow and the enzyme is inhibited. Cells growing concentration of 100mM concentration cannot be used as baits (Criekinge et al, 1999). Toxicity caused by bait can inhibit the growth of yeast (Zhong et al, 2003). Toxicity tests have to be carried out to after the baits are designed. Autoactivation of the baits should be checked before proceeding to the, library screening as nearly 5% of the protein can initiate transcription without an interactor (Criekinge et al, 1999). After the library screening the plasmids can be isolated and used to transform bacterial cells. The interaction also has to be confirmed and isolated by techniques such as coimmunoprecipitation. 2. Aim This study was undertaken as a part of the project by Dr. Claire Palmer in finding novel protein-protein interaction for 5-base pair insert in exon 47 of rat cerebellar Purkinje cell(AF051526). Yeast 2 hybrid system was employed to study interaction. Accordingly two protein baits 5inSER and NLSER were constructed by colleague Surya to screen against library protein. Baits 5inSER is a 472 base pair length protein with ggcag NLSER is a 397 base pair length protein without the Nuclear Localisation Sequence. It was constructed to find the significance of the nuclear localisation signal (Surya, 2008). The aims of the project are To test for toxicity and autoactivation of baits. To determine the concentration of 3-AT at which the expression of Histidine gene is inhibited. Control mating experiment. 3. Materials and Methods 3.1. Control Mating 3.1.1. Control strains The control mating experiments were done prior to the library screen. The positive control yeast strains AH109 with the bait [pGBKT7-53] and Y187 with the target [pGADT7-T] , glycerol stock were provided. For negative control the bait strain was L40 with bait pBTM116/GluR2 and the target was the same Y187[pGADT7-T] The negative control bait was obtained by the transformation of L40 with the plasmids isolated from provided E.Coli cultures. 3.1.2. Small Scale Yeast Transformation A colony of Saccharomyces cerevisiae L40 yeast was inoculated into 10ml of YPAD media. It was left overnight in a shaking incubator (200rpm) at 30à ¢Ã‚ Ã‚ ° C. The overnight culture was diluted in 50 ml of YPAD to an OD600