The PCR amplification of desired genes werecarried out using Taq polymerase reaction kit obtained from Fermentas.Typically, a reaction mixture containing primers with plasmid containing geneof interest or c-DNA was prepared as described in Table 2.6. Table2.6: Various components of PCR reaction mix

Polymerase Chain Reaction (PCR)

and A190middle (557-1100 a.a)fragments were amplified from the plasmid pRS314RPA190gifted byDr. Herbert Tschochner with primers containing BamHI and XhoI (Table 2.4).The mutant versions of A34 and A190 fragments were generated by overlap extension PCR based method. p416GPD GST-RPA43 used in ChIPwas generated by extracting the GST-RPA43fragmentsfrom pYesGex6p2 GST-RPA43by restriction digestion with HindIII and XhoIfollowed by ligation into p416GPD. Clones were verified by bacterial colony PCR, sequencing and western blot. For colony PCR a small amount of bacterial colony was taken with the help of tooth pick and touch the bottom of the 0.2 mL PCR tube to which PCR reaction mix was added and performed PCR

The nomenclature system for RNA Pol I subunits, gene and protein name is given in Table 2.4 and The S.cerevisiaeRNA Pol I subunits Uaf30, A34.5, A43 full length and fragmentsof A135 (1-112 a.a) and A190 (1101-1664 a.a) were PCR amplified from genomic DNA of the wild-type strain using high fidelity DNA Taq polymerase, with primers carrying restriction sites for BamHI and NotI or BamHI and XhoI (Table 2.4). Amplified fragments were cloned downstream of the GAL4promoter in the pYesGex6p2 plasmid (Werneret al., 2010).These plasmids were generated by a colleague,Mr. Unnikannan CP,in the laboratory. Fragments and mutant versions of full length RNA Pol I subunits A34.5 and A43 weregenerated using above plasmids as templates (Table 2.3).A190 N-terminal (1-556 a.a)Calculation of adjusted input-Volume of cell lysate: 500 uL-Volume set aside as input: 10 uL, eluted into a final volume of 40 uL-Volume of lysate taken for IP: 490 uL; eluted into 100 uL after IP, of which 90 uL was eluted into a final volume of 40 uL. Therefore, volume of lysate for IP corresponds to (90/100)*490 = 440 uL (approx. 500 uL)1μLof input sample, and 3 μLof immunoprecipitated sample was taken from40μLof eluted volumes, for q-PCR reaction.The input sampletaken for q-PCR = 1/40x10 uL= 0.25 uLThe immunoprecipitated DNAtaken for q-PCR = 3/40x 500=37.5uLTherefore, the Ct values obtained for input samples (Ct input) were from 0.25 μL out of 37.5 uLoftotal lysateAdjusted Ct=[Ct(Input)-log2(input dilution factor)]The dilution factor forinput sample was 37.5/0.25 = 150Adjusted Ct = [Ct(Input)-log2(150)] = Ct(Input)-7.23Table. 2.6 The gene name and correspondingprotein name for RNA Pol Isubunits.RPA represents RNA polymerase I and protein name starts with A which represents that the subunit is present in RNA pol I

Cloning of S.cerevisiaeORFs

HCT116 cells stably expressing NT or shRNA against IP6K1were grown to subconfluence. Logarithmic phase monolayer cultures were harvested by trypsinization, cell number was determined using a hemocytometer,and resuspended in complete DMEM. Before injection, cells were washedand resuspended in PBS at 2×107cells/mL. Approximately 2×106 cellswere injected subcutaneously into either flank of 6 week old female homozygous Foxn1nuathymic nude mice(n= 8 mice) and tumor size was monitored every 3 days for a period of 4 weeks. Mice were euthanized 4 weeks after injection and tumors were surgically excised and weighed

In vivoxenograft model

The guaranties of the Constitution will then no longer exist; the equal rights of the States will be lost. The slaveholding States will no longer have the power of self-government, or self-protection, and the Federal Government will have become their enemy.

We affirm that these ends for which this Government was instituted have been defeated, and the Government itself has been made destructive of them by the action of the non-slaveholding States.

By this Constitution, certain duties were imposed upon the several States, and the exercise of certain of their powers was restrained, which necessarily implied their continued existence as sovereign States. But to remove all doubt, an amendment was added, which declared that the powers not delegated to the United States by the Constitution, nor prohibited by it to the States, are reserved to the States, respectively, or to the people.

Thus were established the two great principles asserted by the Colonies, namely: the right of a State to govern itself; and the right of a people to abolish a Government when it becomes destructive of the ends for which it was instituted. And concurrent with the establishment of these principles, was the fact, that each Colony became and was recognized by the mother Country a FREE, SOVEREIGN AND INDEPENDENT STATE.

declared that the frequent violations of the Constitution of the United States, by the Federal Government, and its encroachments upon the reserved rights of the States, fully justified this State in then withdrawing from the Federal Union;

14

In this kind of situation one might well ask: why continue to make the 80 per cent of products that only generate 20 per cent of profits? Companies rarely ask these questions, perhaps because to answer them would mean very radical action: to stop doing four-fifths of what you are doing is not a trivial change.

There are two routes to achieving this. One is to reallocate the resources from unproductive to productive uses, the secret of all entrepreneurs down the ages. Find a round hole for a round peg, a square hole for a square peg, and a perfect fit for any shape in between. Experience suggests that every resource has its ideal arena, where the resource can be tens or hundreds of times more effective than in most other arenas. The other route to progress—the method of scientists, doctors, preachers, computer systems designers, educationalists and trainers—is to find ways to make the unproductive resources more effective, even in their existing applications; to make the weak resources behave as though they were their more productive cousins; to mimic, if necessary by intricate rote-learning procedures, the highly productive resources. The few things that work fantastically well should be identified, cultivated, nurtured and multiplied. At the same time, the waste—the majority of things that will always prove to be of low value to man and beast—should be abandoned or severely cut back.

Certainly, the principle brings home what may be evident anyway: that there is a tragic amount of waste everywhere, in the way that nature operates, in business, in society and in our own lives. If the typical pattern is for 80 per cent of results to come from 20 per cent of inputs, it is necessarily typical too that 80 per cent, the great majority, of inputs are having only a marginal—20 per cent—impact.

Both phenomena help to show how the universe abhors balance. In the former case, we see a natural flight away from a 50/50 split of competing phenomena. A 51/49 split is inherently unstable and tends to gravitate towards a 95/5, 99/1 or even 100/0 split. Equality ends in dominance: that is one of the messages of chaos theory. The 80/20 Principle’s message is different yet complementary It tells us that, at any one point, a majority of any phenomenon will be explained or caused by a minority of the actors participating in the phenomenon. 80 per cent of the results come from 20 per cent of the causes. A few things are important; most are not.

Related to the idea of feedback loops is the concept of the tipping point. Up to a certain point, a new force—whether it is a new product, a disease, a new rock group or a new social habit such as jogging or roller-blading—finds it difficult to make headway. A great deal of effort generates little by way of results. At this point many pioneers give up. But if the new force persists and can cross a certain invisible line, a small amount of additional effort can reap huge returns. This invisible line is the tipping point.

We can see positive feedback loops operating in many areas, explaining how it is that we typically end up with 80/20 rather than 50/50 relationships between populations. For example, the rich get richer, not just (or mainly) because of superior abilities, but because riches beget riches. A similar phenomenon exists with goldfish in a pond. Even if you start with goldfish almost exactly the same size, those that are slightly bigger become very much bigger, because, even with only slight initial advantages in stronger propulsion and larger mouths, they are able to capture and gobble up disproportionate amounts of food

At the heart of this progress is a process of substitution. Resources that have weak effects in any particular use are not used, or are used sparingly. Resources that have powerful effects are used as much as possible. Every resource is ideally used where it has the greatest value. Wherever possible, weak resources are developed so that they can mimic the behaviour of the stronger resources.

Why should you care about the 80/20 Principle? Whether you realize it or not, the principle applies to your life, to your social world and to the place where you work. Understanding the 80/20 Principle gives you great insight into what is really happening in the world around you.

The reason that the 80/20 Principle is so valuable is that it is counterintuitive. We tend to expect that all causes will have roughly the same significance. That all customers are equally valuable. That every bit of business, every product and every dollar of sales revenue is as good as another. That all employees in a particular category have roughly equivalent value. That each day or week or year we spend has the same significance. That all our friends have roughly equal value to us. That all enquiries or phone calls should be treated in the same way. That one university is as good as another. That all problems have a large number of causes, so that it is not worth isolating a few key causes. That all opportunities are of roughly equal value, so that we treat them all equally. We tend to assume that 50 per cent of causes or inputs will account for 50 per cent of results or outputs. There seems to be a natural, almost democratic, expectation that causes and results are generally equally balanced. And, of course, sometimes they are. But this ‘50/50 fallacy’ is one of the most inaccurate and harmful, as well as the most deeply rooted, of our mental maps.

The key point is not the percentages, but the fact that the distribution of wealth across the population was predictably unbalanced.

In business, many examples of the 80/20 Principle have been validated. 20 per cent of products usually account for about 80 per cent of dollar sales value; so do 20 per cent of customers. 20 per cent of products or customers usually also account for about 80 per cent of an organization’s profits. In society, 20 per cent of criminals account for 80 per cent of the value of all crime. 20 per cent of motorists cause 80 per cent of accidents. 20 per cent of those who marry comprise 80 per cent of the divorce statistics (those who consistently remarry and redivorce distort the statistics and give a lopsidedly pessimistic impression of the extent of marital fidelity). 20 per cent of children attain 80 per cent of educational qualifications available. In the home, 20 per cent of your carpets are likely to get 80 per cent of the wear. 20 per cent of your clothes will be worn 80 per cent of the time. And if you have an intruder alarm, 80 per cent of the false alarms will be set off by 20 per cent of the possible causes. The internal combustion engine is a great tribute to the 80/20 Principle. 80 per cent of the energy is wasted in combustion and only 20 per cent gets to the wheels; this 20 per cent of the input generates 100 per cent of the output!

The 80/20 Principle asserts that a minority of causes, inputs or effort usually lead to a majority of the results, outputs or rewards. Taken literally, this means that, for example, 80 per cent of what you achieve in your job comes from 20 per cent of the time spent. Thus for all practical purposes, four-fifths of the effort—a dominant part of it—is largely irrelevant. This is contrary to what people normally expect.

Ich habe mal versucht, ein bisschen rauszukriegen, ob denn diese 20 Prozent in der Praxis erreicht werden. Das scheint unterschiedlich zu sein. Aber zum Beispiel beim größten deutschen und auch weltweiten Internetknoten, dem DE-CIX in Frankfurt, liegt die regelmäßige durchschnittliche Auslastung unter 20 Prozent, sodass man davon ausgehen kann, dass der Bundesnachrichten-dienst den DE-CIX komplett überwachen dürfte.

Das G-10-Gesetz sagt, dass die Telekommunikations-überwachung sich maximal auf 20 Prozent der Übertragungskapazität des jeweiligen Übertra-gungswegs beziehen darf.

An effective data management program would enable a user 20 years or longer in the future to discover , access , understand, and use particular data [ 3 ]. This primer summarizes the elements of a data management program that would satisfy this 20-year rule and are necessary to prevent data entropy .