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Is junk DNA bunk? A critique of ENCODE. Edited by Michael B. Law Essay India! Eisen, Howard Hughes Medical Institute, University of California, Berkeley, CA, and accepted by the Editorial Board February 4, 2013 (received for review December 11, 2012) Do data from the Encyclopedia Of DNA Elements (ENCODE) project render the notion of junk DNA obsolete? Here, I review older arguments for junk grounded in the C-value paradox and propose a thought experiment to challenge ENCODE’s ontology. Specifically, what would we expect for the number of functional elements (as ENCODE defines them) in genomes much larger than our own genome? If the number were to stay more or less constant, it would seem sensible to essays over consider the rest of the DNA of larger genomes to be junk or, at least, assign it a different sort of role (structural rather than informational).
If, however, the law essay 2013, number of questions shrew, functional elements were to rise significantly with C-value then, ( i ) organisms with genomes larger than our genome are more complex phenotypically than we are, ( ii ) ENCODE’s definition of competition 2013 india, functional element identifies many sites that would not be considered functional or phenotype-determining by standard uses in biology, or ( iii ) the same phenotypic functions are often determined in a more diffuse fashion in essays by ayn, larger-genomed organisms. Good cases can be made for propositions ii and iii . A larger theoretical framework, embracing informational and structural roles for DNA, neutral as well as adaptive causes of complexity, and selection as a multilevel phenomenon, is law essay 2013 needed. There is much excitement in essay questions the taming of the, the blogosphere, among mainstream science journalists, and within the law essay 2013 india, community of practicing genome biologists about a flurry of fences essay, articles and letters in the September 6th, 2012 issue of Nature . These papers and many others published at about the same time and since under the umbrella of the ENCODE project collectively claim function for law essay competition the majority of the 3.2 Gb human genome, not just the essay the taming of the shrew, few percent already recognized as genes (traditionally defined) or obvious gene-controlling elements. Kolata writes in The New York Times that “[t]he human genome is competition 2013 packed with at least four million gene switches that reside in bits of DNA that were once dismissed as ‘junk’ but that turn out to play critical roles in controlling how cells, organs and other tissues behave” (1). In a Nature News and what do you want from college essay View commentary, Ecker et al. (2) assert that “[o]ne of the more remarkable findings described in the consortium’s entree paper is that 80% of the genome contains elements linked to biochemical functions, dispatching the widely held view that the human genome is mostly ‘junk DNA.’” The editors of The Lancet (3) enthuse: “Far from being ‘junk,’ the DNA between protein encoding genes consists of law essay competition, myriad elements that determine gene expression, whether by switching transcription on computing, or off, or by law essay 2013 india regulating the in gertrude, degree of transcription and consequently the concentrations and function of india, all proteins.” Succinctly, in Science , Pennisi (4) declares that the ENCODE publications write the “eulogy for junk DNA.” The new data—coming from issue paper, high-throughput analyses of transcriptional and law essay chromatin landscapes, transcription factor footprints, and long-range chromosomal interactions—support many current population genetic studies linking human diseases to supposedly nongenic regions, and they are truly impressive in scope and depth (5). They resonate with the paper, current enthusiasm for law essay competition 2013 india assigning multiple subtle but vital regulatory roles to the still enigmatic long noncoding RNAs (lncRNAs) now known to be transcribed from much of the length of our genome (6, 7). Additionally, congruence at many sites between the many methods used (RNA sequencing, binding by issue one or more of 100+ DNA binding proteins, DNase I hypersensitivity, histone modification, DNA methylation, and chromosome conformation capture) leaves no doubt that many of these 4 million gene switches do represent chromosomal loci that are special in some way, in at least one cell type. However, do ENCODE’s data truly require us to competition india abandon the widespread notion that junk DNA—here specifically understood as DNA that does not encode information promoting the survival and reproduction of the organisms that bear it—is the major constituent of many eukaryotic genomes, our own genome included? I will argue by way of a thought experiment and an analysis of what biologists traditionally have understood as function that they do not.
At the very least, “junk” as it has been conceived is an apt descriptor of the computing coursework, bulk of many genomes larger than our own. Moreover, it almost certainly still is for much of our genome, unless we hold Homo sapiens to be unique among the india, animals in the efficiency of its chromosomal organization and not just its cultural attainments. Issue Paper! Such genomic anthropocentrism, unacknowledged conflation of possible meanings of 2013, “function,” questionable null hypotheses, and unrecognized panadaptationism are behind this most recent attempt to junk “junk.” Several of these same points have been made in brief by Eddy (8) and Niu and fences essay Jiang (9). My aim here is to law essay 2013 india remind readers of the structure of some earlier arguments in issue, defense of the junk concept (10) that remain compelling, despite the obvious success of ENCODE in mapping the subtle and complex human genomic landscape. Also, I will suggest that we need as biologists to competition defend traditional understandings of function: the publicity surrounding ENCODE reveals the extent to which these understandings have been eroded. Fences Essay! However, theoretical expansion in other directions, reconceptualizing junk, might be advisable.
The junk idea long predates genomics and since its early decades has been grounded in the “C-value paradox,” the observation that DNA amounts (C-value denotes haploid nuclear DNA content) and complexities correlate very poorly with organismal complexity or evolutionary “advancement” (10 ? ? ? –14). Humans do have a thousand times as much DNA as simple bacteria, but lungfish have at least 30 times more than humans, as do many flowering plants and some unicellular protists (14). Moreover, as is often noted, the disconnection between C-value and organismal complexity is also found within more restricted groups comprising organisms of seemingly similar lifestyle and comparable organismal or behavioral complexity. The most heavily burdened lungfish ( Protopterus aethiopicus ) lumbers around with 130,000 Mb, but the pufferfish Takifugu (formerly Fugu ) rubripes gets by on less than 400 Mb (15, 16). Law Essay Competition India! A less familiar but better (because monophyletic) animal example might be amphibians, showing a 120-fold range from frogs to salamanders (17). Among angiosperms, there is a thousandfold variation (14). Additionally, even within a single genus, there can be substantial differences. Salamander species belonging to Plethodon boast a fourfold range, to cite a comparative study popular from the 1970s (18). Sometimes, such within-genus genome size differences reflect large-scale or whole-genome duplications and essay of the sometimes rampant selfish DNA or transposable element (TE) multiplication. Schnable et al. (19) figure that the law essay competition india, maize genome has more than doubled in issue research, size in the last 3 million y, overwhelmingly through the replication and accumulation of TEs for example. If we do not think of this additional or “excess” DNA, so manifest through comparisons between and within biological groups, as junk (irrelevant if not frankly detrimental to the survival and reproduction of the organism bearing it), how then are we to think of it?
Of course, DNA inevitably does have a basic structural role to play, unlinked to specific biochemical activities or the encoding of competition 2013 india, information relevant to genes and their expression. Centromeres and telomeres exemplify noncoding chromosomal components with specific functions. Essays Rand! More generally, DNA as a macromolecule bulks up and gives shape to chromosomes and thus, as many studies show, determines important nuclear and cellular parameters such as division time and competition 2013 size, themselves coupled to organismal development (11 ? –13, 17). The “selfish DNA” scenarios of 1980 (20 ? –22), in computing coursework, which C-value represents only the outcome of conflicts between upward pressure from reproductively competing TEs and downward-directed energetic restraints, have thus, in subsequent decades, yielded to more nuanced understandings. Cavalier-Smith (13, 20) called DNA’s structural and cell biological roles “nucleoskeletal,” considering C-value to be optimized by organism-level natural selection (13, 20). Gregory, now the principal C-value theorist, embraces a more “pluralistic, hierarchical approach” to what he calls “nucleotypic” function (11, 12, 17). A balance between organism-level selection on competition, nuclear structure and cell size, cell division times and issue developmental rate, selfish genome-level selection favoring replicative expansion, and (as discussed below) supraorganismal (clade-level) selective processes—as well as drift—must all be taken into account. These forces will play out differently in different taxa. Law Essay Competition 2013! Gonzalez and Petrov (23) point out, for instance, that Drosophila and humans are at opposite extremes in fences essay, terms of the competition 2013, balance of issue research, processes, with the minimalist genomes of the former containing few (but mostly young and quite active) TEs, whereas at least one-half of our own much larger genome comprises the moribund remains of older TEs, principally SINEs and LINEs (short and long interspersed nuclear elements). Such difference may in india, part reflect population size.
As Lynch notes, small population size (characteristic of our species) will have limited the do you want from college essay, effectiveness of natural selection in preventing a deleterious accumulation of TEs (24, 25). Zuckerkandl (26) once mused that all genomic DNA must be to 2013 india some degree “polite,” in paper, that it must not lethally interfere with gene expression. Indeed, some might suggest, as I will below, that true junk might better be defined as DNA not currently held to account by selection for any sort of role operating at any level of the biological hierarchy (27). However, junk advocates have to date generally considered that even DNA fulfilling bulk structural roles remains, in terms of encoded information, just junk. India! Cell biology may require a certain C-value, but most of the stretches of noncoding DNA that go to satisfying that requirement are junk (or worse, selfish). In any case, structural roles or multilevel selection theorizing are not what ENCODE commentators are endorsing when they proclaim the end of junk, touting the existence of paper, 4 million gene switches or myriad elements that determine gene expression and assigning biochemical functions for 80% of the genome. Indeed, there would be no excitement in 2013 india, either the press or the computing coursework, scientific literature if all the competition 2013 india, ENCODE team had done was acknowledge an established theory concerning DNA’s structural importance. Questions! Rather, the competition 2013 india, excitement comes from interpreting ENCODE’s data to from college mean that a much larger fraction of our DNA than until very recently thought contributes to our survival and law essay india reproduction as organisms, because it encodes information transcribed or expressed phenotypically in one tissue or another, or specifically regulates such expression. ENCODE (5) defines a functional element (FE) as “a discrete genome segment that encodes a defined product (for example, protein or non-coding RNA) or displays a reproducible biochemical signature (for example, protein binding, or a specific chromatin structure).” A simple thought experiment involving FEs so-defined is at the heart of my argument.
Suppose that there had been (and probably, some day, there will be) ENCODE projects aimed at enumerating, by fences essay transcriptional and india chromatin mapping, factor footprinting, and so forth, all of the FEs in the genomes of Takifugu and a lungfish, some small and fences essay large genomed amphibians (including several species of Plethodon ), plants, and various protists. There are, I think, two possible general outcomes of this thought experiment, neither of which would give us clear license to abandon junk. The first outcome would be that FEs (estimated to law essay competition 2013 india be in the millions in coursework, our genome) turn out to be more or less constant in number, regardless of C-value—at least among similarly complex organisms. Competition 2013 India! If larger C-value by research paper itself does not imply more FEs, then there will, of course, be great differences in law essay competition 2013 india, what we might call functional density (FEs per kilobase) (26) among species. FEs spaced by kilobases in Arabidopsis would be megabases apart in maize on average. Averages obscure details: the extra DNA in the larger genomes might be sequestered in a few giant silent regions rather than uniformly stretching out the computing, space between FEs or lengthening intragenic introns. However, in either case, this DNA could be seen as a sort of polite functionless filler or diluent. At best, such DNA might have functions only of the structural or nucleoskeletal/nucleotypic sort. Indeed, even this sort of competition india, functional attribution is not necessary. There is computing coursework room within an expanded, pluralistic and hierarchical theory of C-value (see below) (12, 27) for law essay india much DNA that makes no contribution whatever to survival and reproduction at fences essay, the organismal level and thus is junk at law essay competition india, that level, although it may be under selection at fences essay, the sub- or supraorganismal levels (TEs and clade selection).
If the human genome is law essay competition india junk-free, then it must be very luckily poised at some sort of minimal size for organisms of human complexity. Of The! We may no longer think that mankind is at the center of the universe, but we still consider our species’ genome to be unique, first among many in having made such full and efficient use of all of its millions of SINES and LINES (retrotransposable elements) and introns to encode the multitudes of lncRNAs and house the millions of enhancers necessary to make us the uniquely complex creatures that we believe ourselves to be. However, were this extraordinary coincidence the case, a corollary would be that junk would not be defunct for many other larger genomes: the term would not need to be expunged from the genomicist’s lexicon more generally. As well, if, as is commonly believed, much of the functional complexity of the human genome is to be explained by evolution of our extraordinary cognitive capacities, then many other mammals of lesser acumen but similar C-value must truly have junk in their DNA. The second likely general outcome of my thought experiment would be that FEs as defined by ENCODE increase in india, number with C-value, regardless of paper, apparent organismal complexity. If they increase roughly proportionately, FE numbers will vary over a many-hundredfold range among organisms normally thought to be similarly complex. Defining or measuring complexity is, of course, problematic if not impossible.
Still, it would be hard to convince ourselves that lungfish are 300 times more complex than Takifugu or 40 times more complex than us, whatever complexity might be. Law Essay India! More likely, if indeed FE numbers turn out to increase with C-value, we will decide that we need to think again about what function is, how it becomes embedded in macromolecular structures, and what FEs as defined by ENCODE have to tell us about it. What do we mean by hamlet in gertrude essay function, informational or otherwise? Most philosophers of law essay 2013, biology, and likely, most practicing biologists when pressed, would endorse some form of the selected effect (SE) definition of in gertrude essay, function (28 ? –30). Selected effect is the form of teleological explanation allowed, indeed required, by Darwinian theory (31). Accordingly, the competition 2013 india, functions of a trait or feature are all and only those effects of its presence for which it was under positive natural selection in over, the (recent) past and for which it is law essay competition 2013 india under (at least) purifying selection now. They are why the trait or feature is there today and computing coursework possibly why it was originally formed. Thus, we might reasonably say that the function of the lac operon in Escherichia coli is (and presumably, long has been) to allow facultative growth of bacteria on law essay competition india, ?-galactosides, because we believe that, long before E. coli was brought into questions of the the laboratory, the lac operon was maintained by selection to allow such growth. We might also say that one of the functions of the human FOXP2 gene (which we share with many other vertebrates) is law essay competition 2013 now to support speech (32), although in the more distant mammalian past, it could not have. We would imagine that there has been selection for speech in human populations over considerable time.
Traits like FOXP2, now under positive or purifying selection for one effect but first arising because of selection for another, are what Gould and Vrba (33) called “exaptations”. What we would not want to call functions (or even exaptations) are effects never so far selected for—side effects, as it were. Gould and Lewontin (34) famously called these “spandrels.” They comprise both undesirable but apparently unavoidable consequences, like vulnerability to phages in bacteria with pili or lower back pain in primates walking upright, and seemingly neutral ones, like the thumping noise made by essays over anthem the heart, to use an example beloved of philosophers. Indeed, even fortuitously advantageous traits, such the FOXP2 -enabled capacity to leave voice messages on answering machines, are not SE functions. India! We do not think that our ancestors experienced positive selection for leaving voice messages, although our descendants well might (and FOXP2 would then for them have acquired a new exaptive function). In any case, past selection, recent or ancient, can only fences essay be inferred, and we must use indirect ways to make the competition 2013, inference. One way, likely the fences essay, most reliable but not universally applicable, is evolutionary conservation.
If diverse lineages retain a DNA sequence despite the erosive force of mutational divergence, there must be some effect maintained by purifying selection. The above is not to say that all conserved sequences are conserved through purifying selection at india, the level of organisms: some may be selfish. Conversely, some conserved functions, such as the complementary base-pairings that maintain ribosomal RNA secondary structures, do not require primary sequence conservation (35). Do You Want College! Moreover, not all sequences that are likely to be currently under purifying organismal selection are conserved on an evolutionary (transspecies) timescale. In a recent comparative genomic survey, Ward and Kellis (36) find both mammal-conserved human sequences showing increasing diversity within our species (and thus, likely becoming nonfunctional in humans) and mammal-nonconserved sequence showing reduced within-human diversity (and thus, likely acquiring new function among us). Ponting and law essay competition india Hardison (37), using methods that they believe to take into account such turnover, “estimate that the steady-state value of ? sel [the proportion of essays anthem rand, all nucleotides in the human genome that are subject to purifying selection because of india, their biological function] lies between 10% and 15%” (37). Another way to attribute function is essays over anthem by ayn rand through experimental ablation: whatever organism-level effect E does not occur after deleting or blocking the expression of a region R of DNA is taken to be the latter’s function. This attribution is close to the everyday understanding of function, as in the function of the 2013 india, carburetor is to oxygenate gasoline. The approach embodies what philosophers would call a causal role (CR) definition of function and fences essay supposedly eschews evolutionary or historical justifications. Much biological research into function is done this way, but I think that most biologists consider that experimental ablation indirectly points to SE. They believe that effect E could, under suitable conditions, be shown to 2013 india have contributed to the past fitness of fences essay, organisms and most importantly, that R exists as it does because of law essay competition india, E . Cardiologists do not say that it is the function of the heart to make a thumping noise, although stopping the heart will silence it.
Similarly, geneticists studying Huntington disease would not say that the trinucleotide repeat in the cognate gene, reiteration of which gives rise to the disorder, has disease causation as a function—although replacing the issue research paper, repeat with a nonidentical set of unique triplets encoding the same amino acid sequence would eliminate the deleterious effect (38). A third, and the least reliable, method to infer function is mere existence. The presence of a structure or the occurrence of a process or detectable interaction, especially if complex, is taken as adequate evidence for its being under selection, even when ablation is competition 2013 india infeasible and fences essay the possibly selectable effect of presence remains unknown. Because our genomes have introns, Alu elements, and india endogenous retroviruses, these things must be doing us some good. Because a region is transcribed, its transcript must have some fitness benefit, however remote. Because residue N of fences essay, protein P is leucine in competition, species A and isoleucine in what do you want college essay, species B, there must be some selection-based explanation. This approach enshrines “panadaptationism,” which was forcefully and effectively debunked by law essay Gould and Lewontin (34) in 1979 but still informs much of molecular and evolutionary genetics, including genomics.
As Lynch (39) argues in his essay “ The Frailty of Adaptive Hypotheses for the Origins of Adaptive Complexity ,” This narrow view of evolution has become untenable in light of recent observations from genomic sequencing and population genetic theory. Numerous aspects of genomic architecture, gene structure, and developmental pathways are difficult to explain without invoking the nonadaptive forces of genetic drift and mutation. In addition, emergent biological features such as complexity, modularity, and evolvability, all of which are current targets of considerable speculation, may be nothing more than indirect by-products of processes operating at computing coursework, lower levels of organization. Functional attribution under ENCODE is of this third sort (mere existence) in the main. India! Although FEs as defined by ENCODE might be cross-identified by several methods and even evolutionarily conserved, they could most often be the computing coursework, molecular equivalent of spandrels—structured elements that are the indirect consequence of selection operating on other features but are themselves selectively neutral, a form of structured noise.
Demonstrations that some biochemical signatures are not neutral and may even meet SE criteria say nothing about the competition 2013 india, rest and are, of course, expected as long as opportunism and co-optation are understood to computing be key elements in evolution. In taking such a liberal definitional course, ENCODE follows the lead of the Gene Ontology (GO) project, which defines molecular function in decontextualized nonhistorical terms (40): Molecular function describes activities, such as catalytic or binding activities, that occur at law essay 2013 india, the molecular level. GO molecular function terms represent activities rather than the entities (molecules or complexes) that perform the actions, and fences essay do not specify where or when, or in competition 2013 india, what context, the action takes place. Proponents of ENCODE actually are concerned with what they call “functional validation” but principally seem worried about the danger of mistaking functional specificity (recognition or activity) rather than the risk of essays by ayn, attributing function (especially regulatory function) where none exists in the SE sense. Stamatoyannopoulos (41) writes: These examples illustrate a natural temptation to equate activity with patterning of epigenomic features. However, such reasoning drifts progressively farther away from experimentally grounded function or mechanistic understanding. The sheer diversity of cross-cell-type regulatory patterning evident in distal regulatory DNA uncovered by ENCODE suggests tremendous heterogeneity and functional diversity.
ENCODE is law essay competition india thus in issue paper, a unique position to promote clearer terminology that separates the identification of functional elements per se from the ascription of specific functional activities using historical experimentally defined categories, and law essay competition also to dissuade the what want college essay, ascription of very specific functions based on a biochemical signature in place of a deeper mechanistic understanding. There are three other “natural temptations” that I would caution consumers of the ENCODE project product to avoid. The first temptation is the assumption that, because some members of a class of law essay 2013 india, elements have acquired SE functions, all or most must have functions or (more broadly) that the class of elements as a whole can thus be declared functional. Stamatoyannopoulos (41), for instance, writes: In marked contrast to essays anthem by ayn rand the prevailing wisdom, ENCODE chromatin and transcription studies now suggest that a large number of transposable elements encode highly cell type-selective regulatory DNA that controls not only their own cell-selective transcription, but also those of neighboring genes. Far from an evolutionary dustbin, transposable elements appear to be active and lively members of the genomic regulatory community, deserving of the same level of scrutiny applied to other genic or regulatory features.
It is surely inevitable that evolution, that inveterate tinkerer, will have sometimes co-opted some TEs for such purposes (42). However, it is an overenthusiastic extrapolation to competition 2013 india describe TEs as a class as “active and lively members of the genomic regulatory community.” Moreover, the do you from, word “regulation” has itself degraded through use by genomicists, from designating evolved effects shown or likely to competition 2013 india enhance fitness, presumably by efficient control of the want college essay, use of resources, to more broadly denoting any measurable impact of one element or process on other elements or processes, regardless of fitness consequences. I think this broadening of definition misleads biologists such as Barroso (43) in competition, a passage cited later in this essay. Pacemakers regulate heartbeats and that is their function: tasers and caffeine also affect cardiac rhythm, but we would not (at least in the former case) see this as regulatory function. Regulation, defined in this loose way, is, for instance, the in gertrude essay, assumed function of many or most lncRNAs, at least for some authors (6, 7, 44, 45). However, the transcriptional machinery will inevitably make errors: accuracy is law essay india expensive, and the selective cost of discriminating against all false promoters will be too great to bear. There will be lncRNAs with promoters that have arisen through drift and fences essay exist only competition india as noise (46).
Similarly, binding to proteins and other RNAs is something that RNAs do. It is issue research paper inevitable that some such interactions, initially fortuitous, will come to be genuinely regulatory, either through positive selection or the neutral process described below as constructive neutral evolution (CNE). However, there is law essay india no evolutionary force requiring that all or even most do. At another (sociology of fences essay, science) level, it is inevitable that molecular biologists will search for and discover some of those possibly quite few instances in which function has evolved and argue that the function of lncRNAs as a class of elements has, at last, been discovered. The positivist, verificationist bias of contemporary science and the politics of competition 2013 india, its funding ensure this outcome. However, what is the correct conceptual framework here? Why should either function or nonfunction for fences essay a class of elements be taken as the null hypothesis, and why should evidence for or against function, however defined, be taken as support of one or the other? Either is law essay competition 2013 india a form of fences essay, essentialism or natural kind thinking inappropriate in contemporary biology.
There is, after all, nothing in nature that constrains classes of genetic elements defined by 2013 india humans as sharing certain common characteristics to share others not part of that definition. The second natural temptation is to research paper assume that function of a part implies function of the whole. Law Essay 2013! Co-optation of the promoters of questions the taming, TEs or the insertion of competition, bona fide regulatory sequences into introns is taken to impart function to the TE or intron as a whole. However, the cell does not necessarily see the fences essay, rest of the TE or the intronic surround of an law essay competition 2013 india, embedded enhancer as relevant to its activity, and only the essays over anthem by ayn rand, promoter or enhancer sequence may be under selection. Even when an entire genetic element seems relevant or necessary (whole introns must be removed even if only certain sites are active in their removal), there is the possibility of excess baggage or junk-like character. Do enhancer-harboring introns really need to be so long? Another analogy seems in order: My computer might be 5 ft from the wall socket, but if I have only law essay a 10-ft electrical cord all 10 ft will seem functional, because cutting the questions, cord anywhere will turn off my machine. In this connection, note that much more than one-half of 80.4% of the human genome that ENCODE deems functional is 2013 so considered because it is transcribed (4), most often into an intron or lncRNA, only a tiny fraction of the length of which is likely to issue be involved in potentially regulatory interactions. A third and related natural temptation is to competition 2013 india inflate functional attribution through choice of window size. The ENCODE Project Consortium notes (5) that: The vast majority (80.4%) of the human genome participates in at least one biochemical RNA- and/or chromatin-associated event in at least one cell type. Much of the genome lies close to a regulatory event: 95% of the genome lies within 8 kilobases (kb) of a DNA–protein interaction (as assayed by bound ChIP-seq motifs or DNase I footprints), and research paper 99% is within 1.7 kb of at law essay 2013, least one of the do you want college, biochemical events measured by ENCODE.
Even this last and largest percentage, assuming the average biochemical event to directly involve tens to law essay competition hundreds of bases (41), assigns functions to only a minority (perhaps 10%) of the genome’s base pairs. Hamlet! ENCODE’s data are indeed unexpected and impressive, but without some principled and law essay 2013 india agreed-on metric for functional density and FE boundaries, any answer to the question “how much of the coursework, genome is functional?” remains endlessly negotiable and transparently window size-dependent. In her News and View editorial in the September 6, 2012 issue of Nature , Barroso (43) speculates as follows concerning the competition, vast majority of human DNA, until now thought useless: …there is a good reason to keep this DNA. Results from the ENCODE project show that most of these stretches of DNA harbor regions that bind proteins and RNA molecules, bringing these into fences essay positions from which they cooperate with each other to regulate the function and level of expression of protein-coding genes. In addition, it seems that widespread transcription from non-coding DNA potentially acts as a reservoir for the creation of new functional molecules, such as regulatory RNAs. In addition to equating regulation with having an effect, Barroso (43) revives here the notion that excess DNA is not junk because it may some day be of competition 2013, use, and issue paper thus it is competition maintained as a reservoir. Brenner (47) long ago derided this sort of essay the taming of the, reasoning as follows: There is a strong and widely held belief that all organisms are perfect and law essay that everything within them is coursework there for a function. Believers ascribe to the Darwinian natural selection process a fastidious prescience that it cannot possibly have and some go so far as to law essay 2013 india think that patently useless features of existing organisms are there as an investment for the future. One sees similar Panglossian futuristic speculation in some of the recent literature on robustness and evolvability (critique in hamlet in gertrude essay, ref. 48).
However, it cannot in general be the case that selection operating at the level of law essay india, fitness of individuals within a species can favor the origin or maintenance of traits that incurs selective cost at that level, while offering only the remotest hope of future benefit to the individual and its descendants. Other evolutionary mechanisms can. Hamlet Essay! The publications by Lynch et al. (24) and Lynch (39, 49) have effectively argued that drift operating in very small populations will (by chance) encourage accumulation of DNA that can add to C-value and law essay 2013 might, in fences essay, the future, come in handy. Selection at the suborganismal (selfish DNA) level may also seem to be future-directed: TEs do sometimes later become useful through co-optation or general effects on the generation of novelty (42, 50). Indeed, Fedoroff (51) has recently proposed that TEs should not be described pejoratively as “selfish” and that the prevailing view—that the competition 2013 india, epigenetic silencing mechanisms that eukaryotes use to limit TE replication arose to hamlet in gertrude essay do just that—puts the evolutionary cart before the horse. Law Essay! Rather, Fedoroff (51) suggests that these mechanisms, by hamlet in gertrude also limiting recombination between repeated TEs (which makes genomes smaller), allow TEs to accumulate, growing genomes and that this was a “…critical step in the evolution of multicellular organisms, underpinning the ability to diversify duplicates for expression in specific cells and law essay india tissues” (51). Fedoroff (51) concludes that On balance, then, the issue research, likelihood that contemporary eukaryotic genomes evolved in the context of competition, epigenetic mechanisms seems vastly greater than the likelihood that they were invented as an afterthought to combat a plague of coursework, parasitic transposons. However, evolutionary explanations of genome structure need not be either/or in this sense, after it is recognized that selection affecting the law essay competition 2013, genome operates at all (including supraorganismal) levels of the biological hierarchy (12, 27). TEs can be selected through selfish replication at what do you essay, the level of DNA, while selection at the level of organisms has established silencing mechanisms to reign in law essay, TE replication, and selection at the level of clades has looked favorably on those clades with complex genomes that engender evolutionary novelty and render whole-clade extinction less likely. What Do You Essay! (That is, clades that have TE-rich dynamic genomes may have indeed because of 2013 india, that produced more and more interestingly diverse and evolutionarily robust and evolvable descendant species.) Evolutionary forces operate simultaneously at all levels in the same and different directions with differing strengths and results measurable in different units (such as the frequency of TEs in an individual genome, TE-enhanced individuals in essay, a species, TE-bearing species in a genus, or classes comprising such species in a phylum). Law Essay Competition India! Indeed, one could reasonably argue for an eventual expansion of the essays by ayn rand, SE definition of “function” to include all levels as long as we distinguish them (DNA-level function, organism-level function, clade-level function, and so forth). This definitional expansion might well lead to a reduction in the amount of DNA that we could reasonably call junk.
However, I think such an competition india, expanded idea of function does not currently inform ENCODE or most genomic thinking, and in any case, the in gertrude essay, problems of law essay, inference, evidence, and appropriate null hypotheses remain. Quite often, we can intuit the results of a thought experiment before articulating the hamlet, reasons for our intuition. Law Essay Competition 2013! This propensity is the want from essay, mysterious appeal and practical use of thought experiments. My intuition is that, when ENCODE-like methods are applied with equal thoroughness to larger genomes, outcomes of the second sort described above will be obtained. That is, lungfish will have many more FEs than Takifugu , and large-genomed Plethodon species will have more than smaller-genomed ones. If there were a primate with a C-value substantially greater than that of H. sapiens , it would prove to have more FEs, even if judged more primitive in intelligence or on behavioral grounds. An exception might be genomes in which C-value increases are very recent and caused by the expansive replication of repetitive sequences that previously lacked ENCODE-definable sites.
Assuming these predictions are borne out, what might we make of it? Lynch (39) suggests that much of the genomic- and systems-level complexity of eukaryotes vis a vis prokaryotes is maladaptive, reflecting the law essay competition, inability of selection to block fixation of over, incrementally but mildly deleterious mutations in the smaller populations of the former. Thus, for india instance, the fact that eukaryotic molecular machines comprise more interacting subunits than their prokaryotic counterparts reflects the inability of questions the taming of the shrew, selection operating on smaller populations to law essay competition 2013 india enforce functional efficiency. Additionally, to be sure, the proliferation of short- and long-range molecular interactions deleteriously interposing themselves within previously simpler regulatory networks will be harder to stop in small than large populations. Several recent publications (52 ? ? –55) have revived the computing coursework, argument that CNE is just such an interpositional force, possibly a very powerful one. A simple example of law essay competition 2013, CNE would be a process by which self-splicing intron RNAs could become dependent on proteinaceous splicing factors. Initially, the questions the taming shrew, RNA secondary structure is sufficient to catalyze self-removal, but fortuitously bound proteins that stabilize the competition, RNA can compensate for (presuppress) mutations that might destabilize elements of the structure necessary for independent splicing. The Taming Of The Shrew! Because they are not now deleterious, such mutations will accumulate to equilibrium: the purifying selection pressure that maintained RNA secondary structure has gone. Law Essay Competition 2013 India! No selection is involved at this stage. If there are several such potentially presuppressible mutations, then a ratchet-like mechanism will make it difficult or impossible for the RNA to essays by ayn ever regain splicing independence. Elimination of the protein will become a lethal event.
Therefore, purifying selection now prevents the loss of the complex feature (molecular interdependency), although positive selection did nothing to create it. The entire multiprotein, multi-RNA, eukaryotic spliceosome might have evolved through reiterations of this process along with very many of the intricacies of the cellular machinery (52 ? ? –55). CNE would work in concert with any population-size effect. Law Essay 2013! Neither entails positive selection for the complex structure and/or processes thus produced—only purifying selection against its elimination. Philosophers who endorse SE definitions of function have not, to my knowledge, embraced or even considered such CNE scenarios, which would meet CR definitions. Considering traits fixed by issue paper CNE to have SE function would add still additional arrows to the quiver of panadaptationism and law essay 2013 should perhaps be discouraged. A common consequence of questions the taming of the, CNE is that even structures or processes that have arisen by positive selection because they increase organismal fitness will later become more complex in terms of the number of intermolecular interactions required for their successful completion. Function diffuses. Law Essay 2013 India! Genetic networks will first acquire and then require more and more protein–nucleic acid, protein–protein, and nucleic acid–nucleic acid intermolecular associations. Do You Want College Essay! Larger genomes, producing more RNAs (and sometimes more proteins), offer up more macromolecules and variants as potential fortuitous presuppressors and more potential DNA binding sites.
Recognition systems for transcription and transcription factor binding cannot be made indefinitely more accurate without the aid of unbearably slow and selectively costly proofreading systems. Tradeoffs between speed, economy, and accuracy will unavoidably entail that larger genomes will produce disproportionately more noise in terms of competition, fortuitous transcripts capable of becoming presuppressors and thus, more complex, seemingly regulatory, networks of in gertrude, interaction. Some will be functional in a CR if not an SE sense, and some will have arisen through CNE so that they are now maintained by purifying selection. However, many, possibly the law essay competition, vast majority, are just there. The above considerations are but some of the hamlet essay, reasons that one might intuit that FE number will scale with genome size. A very recent comparative analysis of transcription factor binding sites in law essay competition, model organisms (45) confirms this conjecture.
Ruths and shrew Nakhleh (45) claim that …neutral evolutionary forces alone can explain binding site accumulation, and that selection on the regulatory network does not alter this finding. If neutral forces drive the accumulation of binding sites, then, despite selective constraints, organisms with large amounts of [noncoding] DNA would evolve functional, yet ‘overcomplicated’ networks. The renewed debate over junk, thus, owes much of competition, its heat to want from essay at least four misconceptions or misrepresentations. First is the pretense that there is any definable boundary between informational and structural (genic and nongenic) functions for DNA. Increasingly, genomics is expanding the boundaries of information as geneticists have typically understood it. Competition 2013 India! Minimally, gene means more than it used to computing mean. Djebali et al. (56) write …the determination of law essay india, genic regions is currently defined by the cumulative lengths of the hamlet essay, isoforms and their genetic association to phenotypic characteristics, the likely continued reduction in law essay india, the lengths of intergenic regions will steadily lead to the overlap of most genes previously assumed to be distinct genetic loci. This supports and is consistent with earlier observations of a highly interleaved transcribed genome, but more importantly, prompts the reconsideration of the definition of a gene. Want Essay! As this is a consistent characteristic of annotated genomes, we would propose that the transcript be considered as the basic atomic unit of inheritance. Concomitantly, the term gene would then denote a higher-order concept intended to capture all those transcripts (eventually divorced from competition 2013 india, their genomic locations) that contribute to a given phenotypic trait. However, regulatory loci are also informational even if not transcribed, and computing ENCODE has documented many long-range interactions between chromosomal regions that may be brought together physically in the nucleus, a very complex and structure-rich molecular machine, at some time during the cell cycle.
Therefore, in this sense, the gross structure of the chromosome set also carries information that may be relevant to the function of genes, broadly defined. Competition 2013! Additionally, all DNA has the job of essays over by ayn rand, serving as a template for competition 2013 its own replication—to that extent, encoding information. It is nevertheless true that a distinction between structural and informational roles has long been part of the C-value argument for over anthem rand junk DNA. Competition! This line of reasoning has held that high C-value might be necessary for cellular function, but the computing coursework, nongenic DNA that fills the requirement is law essay 2013 india informationally junk. ENCODE’s claim is that much more of the DNA is, in fact, informational (especially regulatory) than we had thought, and indeed ENCODE’s focus is on sites likely to want from essay be involved directly or indirectly in transcription—on the “myriad elements that determine gene expression” to quote The Lancet (3). Therefore, the structure–information distinction informs the interpretation of the project’s results, and without it there would be nothing novel or newsworthy in law essay competition india, the assertion that all of the human genome has some sort of anthem by ayn, role in human biology. We have known that since the law essay, mid-1980s.
Second is the essays over rand, conflation of SE and CR definitions of function. Those of us who speak of excess DNA as informationally junk mean that its presence is india not to be explained by past and/or current selection at the level of organisms—that it has no informational function construable historically as an SE. Those who say that almost the whole of the human genome is functional informationally do so on the basis of an operational diagnosis embracing a nonhistorical CR definition of function. This definition is issue research paper certain to identify as functions very many effects that have not been selected. The rhetoric attending the declared “eulogy for junk DNA” (4) sweeps this distinction under the carpet. Third is law essay a false natural kind ontology, essentialist in fences essay, nature, that encourages ( i ) the attribution to a whole class of operationally defined genetic elements those functions known only for a few and/or ( ii ) the law essay competition 2013, attribution to the whole length of such a genetic element a function that resides in only part of it. Fences Essay! In the 2013 india, case of lncRNAs and issue intron transcripts, whose lengths together make up more than three-quarters of 80% of the genome said to law essay competition 2013 india be functional, this second sort of functional attribution seems especially misleading. Fourth may be a seldom-articulated or -questioned notion that cellular complexity is adaptive, the product of fences essay, positive selection at the organismal level. Our disappointment that humans do not have many more genes than fruit flies or nematodes has been assuaged by evidence that regulatory mechanisms that mediate those genes’ phenotypic expressions are more various, subtle, and sophisticated (57), evidence of the sort that ENCODE seems to vastly augment.
Yet there are nonselective mechanisms, such as CNE, that could result in the scaling of FEs as ENCODE defines them to C-value nonadaptively or might be seen as selective at some level higher or lower than the level of 2013 india, individual organisms. Splits within the fences essay, discipline between panadaptationists/neutralists and those researchers accepting or doubting the importance of multilevel selection fuel this controversy and others in biology. I submit that, up until now, junk has been used to denote DNA whose presence cannot reasonably be explained by competition 2013 natural selection at questions of the, the level of the competition 2013 india, organism for coursework encoded informational roles. Law Essay! There remain good reasons to research paper believe that much of the DNA of many species fits this definition. Nevertheless, while still insisting on SE functionality, we might want to come up with new definitions of function and junk by ( i ) abandoning the law essay 2013 india, distinction between informational and coursework nucleoskeletal or nucleotypic roles for DNA, ( ii ) admitting that there may be strong selection for C-value as a determinant of many cell biological features, ( iii ) fully embracing hierarchical selection theory and acknowledging that different genomic features may have legitimate functions defined and in play at different levels, and ( iv ) expanding the SE definition of function to include traits that arise neutrally but are preserved by law essay 2013 india purifying selection (12). Much that we now call junk could then become functional. However, such a philosophically informed theoretical expansion is not what ENCODE, or at least those authors stressing the demise of research paper, junk, so far seem to have in mind (1 ? ? ? –5). In the end, of course, there is india no experimentally ascertainable truth of these definitional matters other than the truth that many of the most heated arguments in essays rand, biology are not about facts at all but rather about the words that we use to competition describe what we think the facts might be. However, that the computing coursework, debate is in the end about the meaning of words does not mean that there are not crucial differences in our understanding of the evolutionary process hidden beneath the rhetoric. I note that a very forcefully worded critique by Graur et al. (58), with more specific objections to ENCODE's methodology, was published while this manuscript was in the proof stage.
I thank Evelyn Fox Keller, Eric Bapteste, James McInerney, Andrew Roger, and the Centre for Comparative Genomics and Evolutionary Bioinformatics (CGEB) group for comments on this manuscript and law essay 2013 india the Canadian Institutes for what do you from Health Research for support.
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5.0 Marketing Strategy and Implementation. THR will position itself as the 2013, premier home-style restaurant in the Benbrook suburb of Fort Worth, Texas. We will do this by providing quality home style meals, prepared with quality ingredients at what college essay, a reasonable prices. 2013! Customers will enjoy the quaint surroundings inside with the fences essay, wood tables and checkered table cloths. Competition 2013! Our restaurant will provide a relaxed atmosphere and when customers walk in they will be greeted by hamlet essay, warm smiles and greeted just as they were arriving home. The chains have tried to create home-style restaurants but where they have failed is in the personal aspect of the business. The POS system known as “The Expediter” used to competition 2013 india, monitor inventories and time meals has replaced one of the most important aspects of a restaurant – the friendliness of the staff! And in light of this, a handful of chain restaurants are beta testing self-pay tables! Our customers will enjoy our standard menu fare, along with seasonal menus so that we can better take advantage of cost savings and stay current with some of the food industry trends. At THR we plan to be the premier restaurant to work for essay questions the taming shrew, as well.
We believe that the restaurant industry is a great place to begin one’s career or pursue full-time. In fact, according to the Restaurant Association, nearly half of all adults have worked in the restaurant industry at some point during their lives, and more than one out of 2013 india, four adults got their first job experience in a restaurant. At THR we believe that our restaurant will provide job opportunities both for the entry level applicant was well as for the part-time worker searching for flexibly in job hours. Issue Research Paper! We will pay our employees a competitive salary and believe we can do so by meticulously keeping our records, including daily review of the 2013 india, Prime Cost Report, and utilize Cost Accounting Systems, to prevent inventory shortfalls. We will be proactive with our employees by scheduling regular performance reviews, and in gertrude, provide bonuses and 2013 india, other incentives to motivate our staff. Computing Coursework! We will also provide our employees with the law essay competition 2013, most current training programs regarding safe food handling, and worker protection. (Additional information regarding our employees is explained in computing coursework, Section 6.1 which follows).
THR will also remain current with current industry marketing tends. Law Essay 2013! In addition to a website with our menu, map and driving directions, we will also have a Facebook page and do you want from college essay, utilize other social media such as Twitter. We will team up with Groupon and law essay 2013 india, Yelp. We will offer a loyalty club and birthday club which recent reports indicate increases earnings as much as 15%. Prime location with easy access from Interstate 30 Exceptional staff with the essays over anthem rand, can do attitude. Combined 25 years in the restaurant industry Because owner has catering industry experience, he already has established a customer market and india, approved vendors Due to our small size, we believe we can provide exceptional quality by hand selecting our market specials when compared to our larger corporate competitors The same concept holds true in our staffing requirements, by hand selecting our employees we will strive to offer unsurpassed service when compared to do you, our larger competitors. Recruiting and retaining quality employees Tight margins will allow little wiggle room for error. Little barriers to entry allows for immediate business opportunities Offer additional catering services. Government mandates (restaurant operation, food safety, and worker protection at india, the federal level and health, sanitation, safety, fire at the local level) Rising operating costs Building/maintaining sales volume Supermarkets and convenience stores Consumers that believe that meals at home are healthier than those prepared in restaurants.
Strategy: Be the Benbrook area’s premier Home-Style Food Restaurant in Customer Satisfaction. Tactics: First create awareness-- our signage on want college essay the front of restaurant will bring customers to us and once inside, we will immediately acknowledge the customer with the warmest and most sincere greeting and begin the service process anticipating repeat customers. Programs: Provide employee training on customer service and retention; offer ongoing training programs for employees keeping them current on industry trends and food safety. Law Essay 2013! Keep track of employee’s progress through performance reviews and offer employees incentives attracting and what want from college essay, retailing customers. Employ Mystery Shoppers. Employ the use of surveys both at the table and online. 5.3 Unique Selling Proposition (USP)
THR will be able to offer home-style meals for a reasonable price in a comfortable ‘home-like’ setting. The average check price is expected to be between $8-$15 which appears in line with industry standards below $25.00 (First Research). Because of competition 2013 india, our current expertise with vendors, and our excellent credit, we can negotiate better credit terms than say someone brand new starting a restaurant. We will also be able to keep our menu reasonably priced by offering menu items that take advantage of issue, seasonal produce further reducing price. Finally we will keep our prices in check by meticulous monitoring of our controllable expenses – keeping close eye on our Prime Cost Report and Inventory. By initially employing family members who will work for lower and law essay 2013 india, reduced wagers, for in gertrude essay, example, we can further reduce our controllable expenses. THR’s competitive edge is in its people. We truly believe that your business is not only as good as your products (meals) but the quality of your staff as well. Our staff is a reflection of law essay 2013, us.
Initially, we intend to employ our family members who will work for hamlet essay, lower and reduced wages. Our long term goal is to hire team members that are truly hand selected and have the same honest to goodness family values we do. And unlike our big chain competitors, because of our lean size, we can turn on a dime when economically pushed and make changes quickly allowing us to be proactive. (Whereas our corporate competitors have to adhere more closely to competition 2013 india, their company policies thus impeding their reaction time) 5.5 Marketing Strategy and Positioning. We realize the success of THR will have to issue research, be achieved by doing more that serving great food, and providing friendly service. We will utilize a marketing plan to build customer traffic. At THR we will continually strive to win more customers by being proactive rather than reactive in our marketing efforts and stay current with popular industry trends. We will achieve these goals by law essay competition 2013, using the following:
Database: We will begin our campaign by marketing to our existing database of essays anthem, customers. We will email fliers announcing our grand opening. We will continually update our database by providing a fishbowl for business cards in the lobby and offer a weekly or monthly drawing. Loyalty Program/Birthday Program. THR will offer a birthday/loyalty club proving a complimentary hamburger or chicken sandwich or wrap to the for the birthday person. A recent report from the National Restaurant Association explained how this simple technique can increase revenues as much as 15% due to repeat business. Competition India! Our restaurant team will also be active in the local community and we plan to take an active role by participating, sponsoring, and donating to local churches, sports clubs or teams in hamlet in gertrude essay, the market area. We will also strive to develop rapport with local business as a quick, comfortable lunch choice.
In the india, future, we plan on establishing a marketing campaign to call on the local business in the market area, deliver samples, and encourage them to consider our restaurant as the issue research, restaurant of choice for their next business luncheon. THR will be the premier home-style dining restaurant in law essay 2013 india, western Fort Worth. Questions Of The Shrew! We will offer reasonably priced meals, in a warm, relaxed and comfortable setting. We have a wide selection on our menu and also have menu options for lighter fare as well as a children’s menu. We are open 7 days a week and unlike our chain competitors, our servers won’t try to be your best friend our rush you thought your meal. Our name says it all “Traditional Home-Style Restaurant - - honest to goodness food served to you by honest to competition 2013, goodness people!” At THR, cost accounting is important, since the issue paper, profitability of individual dishes can vary significantly and will initially determine the cost of the 2013, menu items. We will take advantage of essays over anthem by ayn, our excellent credit terms with our suppliers and will also update our menu to law essay competition 2013, take advantage of seasonality for example in local produce items.
We will also closely monitor the Prime Cost Report which focuses on the controllable expenses of Cost of Goods Sold and Labor. As a new start-up we can currently control employee cost by fences essay, hiring family members who will work for low and reduced wages. 5.5.3 Promotion and Advertising Strategy. Location- The restaurant will be located in competition india, a strip center at fences essay, the busy intersection of Cherry road and Camp Bowie. Law Essay 2013! With easy access to Interstate 30 (less than ? mile south) and located on the “going home side” (Western Corner) of the intersection. We will have Signage on the West Roads Shopping Plaza as well as signage over questions shrew our entrance Word of Mouth – We already have a database of existing catering customers and will rely heavily on this method to attract and grow new business.
Participate with Costco as Small Business of the Month – We will leave our menu, a fishbowl for business cards and a small ‘homey’ display with the competition, retailer announcing us as new entrants in hamlet essay, the local restaurant arena. Direct Mail - Bulk mailing either directly to potential customers or by law essay 2013 india, including a postcard in a value-pack-type mailing. Essay! Event Marketing -We plan on joining our local chamber of commerce and utilizing their networking services for our grand opening. We will stay current with industry trends and have a webpage, Facebook page and law essay 2013, Twitter site. Our menu, map, and hours of operation will be easily accessed.
In the future we may consider fax or email orders as well a phone application. Our initial marketing campaign will consist of contacting our databases clients and notifying them of our grand opening. Fences Essay! We will seek the use of law essay competition india, a local mailing service program to assist us in the implementation of the campaign. Ongoing- we will meticulously keep our database current and hamlet, use the Constant Contact program Loyalty and law essay 2013 india, Birthday Club members will notified of upcoming special menu items and to alert them of our catering service. Customer service is of the fences essay, utmost importance. Customer surveys estimate that only 1 in 20 customers that have a problem in a restaurant will tell management about it. Law Essay! It will be our goal to provide a wonderful home-style meal combined with superior customer service.
Training programs will include teaching materials to train our employees about service attitudes, customer perception and how to handle guest complaints. Jeff and Betty will conduct periodic staff meetings intended to review policy, increase guest satisfaction and to fences essay, keep a general line of communication between staff and management. All guest complaints will be acknowledged by the staff and referred to management. Law Essay Competition 2013! Programs will be in research paper, place for all types of guest complaints. Law Essay 2013! More serious complaints will be documented and kept on file. Customer feedback will be accomplished by customer surveys or the use of mystery shoppers. We are expecting a conservative 5% increase in sales revenues annually over the next 3 years. The growth is fences essay, adjusted for inflation. With the addition of catering revenues, sales will increase by law essay 2013 india, 12.93% in Year 2 and 6.02% in Year 3. The following table shows expected Sales Forecast for the next 3 years: Table 5.6.1 Annual Sales Forecast.
We will encourage our employees to grow our customer base and provide incentives and regular bonuses to employees for over, referrals and repeat customers. India! These initiatives are still in the planning stages as we gear up to hire and staff. Coursework! They will play an law essay india active role in our employee culture. It is also anticipated that as we grow our catering business, along with our lunch business group, we will hire a sales director to in gertrude, facilitate this portion of the business. The sales director will be compensated similarly to their national peers (national Restaurant Association) Initially we will be formed a sole proprietor: Jeff Wright d/b/a Traditional Home Style Restaurant. The State of Texas is a community property state.
Over time, the competition 2013, couple plans to issue paper, form a Limited Liability Company. Our initial milestones are as follows: Table 5.8 Milestones. THR expects to hire 19 employees. Together, Jeff and Betty Wright will personally select each candidate. They’ve adopted an effective interview process designed to staff the restaurant with highly qualified people for each position. Each applicant will be rated and evaluated according to a pre-defined set of standards designed for each position. Background checks will be utilized for designated positions. Recruiting efforts will always center on referrals. The restaurant will be owned by Jeff Wright.
Jeff began his restaurant career at the age of 15 working in india, a quick-service foodservice operation and earned his way through college as a server and bartender. After earning his degree, he worked for a regional restaurant chain and what do you want from college essay, an independent fine dining restaurant. In these organizations he held the law essay 2013 india, positions of Assistant Manager and then General Manager. Betty Wright received her Culinary Degree from the Art Institute in Dallas. After graduation she was employed by a local chain restaurant and then at issue research, a Five Star Hotel in Dallas. Betty will initially be employed as the Kitchen Manager. Initially Jeff and Betty will fill in many of the management gaps. Over time, they have plans to law essay india, hire a sales director, a general manager, and a kitchen manager.
To meet the gaps associated in payroll, inventory management, and cost accounting, the essay questions the taming shrew, Wrights will purchase have considered a POS (point of sale system) that it simplifies communications between the kitchen and the wait staff. Orders go through the competition india, computer, directly to the kitchen printer. Another benefit of a restaurant POS programs is that it can track everything from food usage, to the most popular menu items. Because the essays anthem by ayn, POS system acts as a time clock, it can also help prepare payroll – which will save some money in the bookkeeping department. Along with the daily operations of running a restaurant, a POS system can organize profit and loss statement and sales tax.
Table 6.4 Personnel Plan. The following sections outline our financial plan: Required Cost of Start-Up Profit and Loss Cash Flow Balance Sheet Financial Ratios Hourly Labor Costs Weekly Sales Projections. Meal Price range from $8.00 - $15.00 Average lunch price: 8.79 Average dinner price: 13.74 The restaurant is located in the West Roads Shopping Center and is comprised of 3,400 square feet The dining room will be comprised of law essay competition india, 20 tables with a seating capacity of 86 seats and hamlet in gertrude essay, 40 available parking spaces to meet the needs of the customers. The restaurant will employ 19 employees $860,000 -1,200,000 revenue target; Industry average for casual restaurant average of law essay competition 2013 india, $860,000.
Annual 3% increase for inflation and 5% annual increase in fences essay, revenues Year 2 Assumes Catering Business in Place. Assumes 4 parties monthly @ $15 per plate and $50 persons. Catering will escalate to law essay 2013, 8 parties monthly in do you, month 20 and then 10 parties monthly thereafter. Law Essay Competition! Also assumes additional increase in staffing (4 persons to be hired at 6 hours @ $8.00 per hour. Total start up costs will be $363,000, $174,000 of which will be contributed by the owners and the remainder will be secured through a proposed bank loan. Table 7.2 Start-Up Costs. Total start-up costs are estimated to be $363,000. The majority of the costs are associated with the restaurant equipment, inventory and furniture and furnishings for anthem by ayn, the dining room.
Total costs for competition 2013 india, these items are reported to be $110,500. What Do You From College! The costs are associated with build out and law essay competition 2013, renovation of the restaurant to provide updated plumbing and essays over anthem, creating additional space in the dining area by removing a non-supporting wall: $50,000. Additional start up expenses are in the form of working capital and contingency $182,500. Jeff and Betty Wright will contribute $174, 000 and are requesting an additional $189,000 in the form of a bank loan. The loan is expected to be a fully amortizing 5 year term note secured by UCC filings on all furniture fixtures and equipment. Table 7.3 Source and Use of Funds. Total fixed costs associated with the restaurant are $669,186 and represent the annual expenses. The variable cost (overhead) is competition, estimated to do you want college, be $4.51 per meal. Based on law essay india the assumption of $11.37 as the average meal price, the breakeven revenue then is $1,108,970 or 97,535 meals (units). This is issue, further depicted in the Table Below and the Graph that follow: Table 7-4 Break-Even Analysis.
The profit and loss demonstrates modest increases in competition 2013, revenues over hamlet the three expected years with adjustments for inflation. Table 7.5.1 Pro Forma Profit and Loss. The statement of cash flow shows the incoming and outgoing cash of the business.
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chapter 5 homework For those who have reviewed, added and law essay competition india favorited this fic. I Won't Thank you. Because Thank you is issue research not enough for your wonderful and awesome response for this story. Whoever knows of a word greater than or more wonderful than 'Thank you' then please inform me of the word. Thank you. Uurgh, I just said I won't Thank you!
THANK YOU VERY MUCH AND I LOVE YOU ALL! ^O^ Somewhere in Karakura town, two high school girls kneel on top of a small hill. silently praying. One girl has a black shawl over her head like a veil, while the other one clad her own black shawl around her shoulder. Competition India. On a blanket before them lies a picture of a random woman, a bottle with a butterfly inside, a pile of stones and pebbles, an incense smoking lightly, and 3 red balloons tied up in a rabbit figurine and other random stuffs. After a moment of silent prayer, one girl opens her eyelids revealing that purple irises of her. The girl next to her did the same and essays anthem rand they clap their hands twice and bow their heads before slowly sitting upright and nodding at each other.
Rukia stands up and grabs the competition 2013 india balloons while Momo gets the incense before joining her friend. They stand next to each other on the edge of the over by ayn rand hill overlooking half of the competition 2013 Karakura town; both are wearing solemn and serious faces. Momo holds the incense on both hands while Rukia holds up the balloon on one hand while the fences essay other holds the hem of her shawl together. Competition. She takes in a deep breath and looks up at in gertrude the sky before she starts, It has been 15 years of joy, sorrow, happiness, pain. and arcade games. Momo sniffs gently, brushing a tiny hint of competition 2013 india tear on the corner of her eye in a very lady like manner, while Rukia resumes on her speech. It has been 15 wonderful years of youth and carefree living. But tonight. will be the end of those years.
But though shall not feel sadness cause the end is computing coursework just the beginning. Competition India. A cold and gentle late afternoon breeze blows around them, rustlings the leaves and computing the grass and making the birds fly off the trees. Tonight, I will surrender my virginity to my 'wonderful' husband- Rukia turns at her friend as the said girl rubs her nose and wipes it with her shawl before mumbling a 'Sorry.' and urging her to go on. She rolls her eyes before taking in a deep breath and holding up the balloons on law essay 2013 her hand. Fences Essay. Let these red balloons symbolize my virginity that I will whole heartedly give to my husband, while these incredibly wonderful rabbit figurine brought us prosperity. Let these fly off towards the sky, and into the moon and law essay competition 2013 be with the stars. And may it reach the fences essay heavens, to send us the angels of love and. Law Essay 2013. Love making. Issue Paper. so Ichigo and I will be prosperous and always happy. And satisfied, Momo murmurs making her friend blush.
Rukia shoots her a quick glare before clearing her throat and law essay competition india holds the balloons higher. Issue Paper. Both of them look up at the sky, It is time. Law Essay Competition 2013. Goodbye virginity! Goodbye. she declaims dramatically as she releases the balloons on her hands. The two girls watch the over by ayn rand red balloons with solemn anticipation as it floats up a bit. and slowly falls down the ground in front of them. I told you to buy the white ones. So, here are the checklists I made for The Preparation.
You have to 2013 follow all of this so. Momo's voice trails off as she displays each item on her bag while Rukia scans the said checklist. Place a photo under his pillow? Rukia reads in a questioning tone and Momo, still busy with the 'stuffs', nods at her. It's for fences essay, dream purposes. It's very effective.
When you place your photo under his pillow, he will be dreaming of you every night. And that'll set his mood, Momo explains. Rukia raises an eyebrow as she continues, Spray or display honey scented fragrant around the competition house. You know, it's for hamlet in gertrude, what they say 'the birds and the bees'. Law Essay Competition 2013 India. It'll attract them. Rukia gives her an unsure look, Always tie up your hair. Sing jazz songs while taking a shower. Momo chuckles, It's very daring and seductive but it's a turn on for sure. But Momo, I don't know jazz.
Oh! Don't worry, I'm prepared. Coursework. Here. Momo hands her a cd. Fly me to law essay 2013 india the moon or Unchained Melody. Oh! What a classic. You have to learn it. Rukia lets out a groan and gives her friend a look of fences essay disbelief. Momo pauses on arranging her display and looks back at her friend. Rukia, it's necessary.
You have to do all of this so you are a hundred percent prepared for competition 2013, the big night of your life! Rukia sighs, But Momo, I- Ah, ah, ah! No more protests, Mrs. What Want College. Kurosaki. You will follow that checklist and that's final. Remember what they say, no pain no glory. You mean, no pain no gain, Rukia deadpans but her friend waves her hand dismissively, mouthing the competition 2013 india word 'Whatever' before continuing on her task.
Rukia sighs and scans the paper again, reading it silently reacting only on a particular one. Lingerie? Momo, why do I have to buy a lingerie? Momo chuckles again at her friend's reaction, Oh that's my favorite. You'll wear it for the lap dance. Rukia blushes and gapes at do you from college her friend, What? You're a dancer, Rukia so. oh! Yeah, I forgot to write there. You also have to buy painkillers, Momo trails off, ignoring her gaping friend.
You know, Kurosaki-sensei sure looks big and he's an india athlete too so he sure has a lot of stamina and. Research. Blood oozes down Rukia's nose and law essay competition her face is red hot as her expert friend continues to talk about anthem by ayn rand Rukia's husband and law essay 2013 how his 'big thick sword' will cut and thrust her 'fresh and young flower'. Seriously, is essay questions the taming shrew Momo still a virgin or she just reads or googles too much? . oh! You will surely cry for his name and asks for him to do it again. Uuuugh. Rukia groans as Momo starts dramatizing beside her. Oh yeah! That's it. As the day's move along, Rukia and Momo busy their selves with The Preparation.
Buying these and competition those items, doing weird stuffs like sitting on the rooftop during breaks doing yoga or some meditaion, catching butterflies in the morning and releasing it before the do you want college essay day ends, Momo always tie Rukia's hair in different styles. They also do this weird dance/exercise and law essay competition Momo even manage to essays over by ayn rand make Rukia wear something along with her uniform. What are you were wearing? Ichigo asks with a raise brow as he looks at his wife up and down. It's called a school uniform.
Rukia says, smiling sheepishly at him. I know it. India. What I mean is. why are you wearing jogging pants under your skirt? It's not the proper school attire. Ah, it's because. ahm. Rukia, it's for the purification and cleanliness. Coursework. Your body should be kept from other people's eyes so you have to wear our coat and law essay competition india wear pants under your skirt. Don't worry it's only for hamlet in gertrude, The Preparation.
You can take it off after your 'honeymoon'. Ahm. It's for PE class later, Rukia answers, a pink blush tinting on her cheeks as she remembers the real purpose of 2013 india it. What College. as what Momo says. But PE is after lunch break so you still have time to law essay competition india change. Rukia, remove those pants.
I don't want you having caught by the Discipline office again so- No! Rukia exclaims. I mean I want to issue research paper wear it now so I'm not taking it off. Don't start at me, Rukia. You know you better take that off or I'll make you. Rukia blushes upon seeing the malicious grin on her husband's face.
But the law essay competition india stubborn part of her kicks in and gives him a glare. Don't you dare. Their eyes narrow at each other and they poses on computing coursework a defensive stance as they walk in competition 2013 india, circles, then. Rukia bolts towards the front door of their house and manages to open it but of course Ichigo, being a fast demon he is, appears right behind her and slams the door shut with one hand. He then lifts his small wife and coursework shoves her on the couch. Rukia groans and sits upright but before she can get away, Ichigo stands in 2013, front of her and orders her firmly. In Gertrude. Take it off. Now. Momo skips happily as she enters the gate and heads towards her friend's house. Earlier this morning, she felt like walking with her bff to school so here she is now. standing in competition india, front of the Kurosaki house and what from essay is about to knock on law essay india its door. OHAY-hm?
Her hand stops mid air as she hears a yelp. Curious, Momo presses her ear on the wooden door and her eyes widen at what she hears. Ichigo, stop it! I'm not- Take it off, Rukia or I'll tear it down myself! No! Please, don't take it away from me! I'm not prepared yet! You don't need any preparation for this, so c'mon Rukia! We still have to go to computing coursework school. Uurggh.
Rukia, you're grabbing it so tight! Aah. Ichigo let me go. you're hurting me! Momo can't take it anymore. She bursts inside the couple's house, screaming. RUKIA! I'LL SAVE YOUUUU. The couple turns at door and india sees a fuming Momo coming their way with a vase on her hand, ready to throw it at. Hmm? Momo pauses on her track, blinking as she finds her raven haired friend sitting comfortably at the couch while her orange haired husband stands on hamlet in gertrude essay the side, a pair of jogging pants on his hand.
Rukia, I thought you. Did he. I mean, how'd you. Law Essay Competition 2013 India. Momo stutters. Rukia frowns and crosses her arms in front. Momo, he won't allow me to fences essay wear it. Ichigo grimaces, realizing that it's Momo's idea after all and tosses the law essay competition 2013 pants aside. Computing Coursework. He then turns at his wife's bff and law essay 2013 india scowls, Why are you holding up a vase, anyway?
Momo, who is still on her throwing stance, turns beat red as the Kurosaki couple stares at her. Hamlet Essay. She holds the vase to her face and competition smiles sheepishly, Roses are really red, aren't they? Walking side by side, Rukia and Momo stares at the orange haired teacher walking a few meters ahead of them. Momo's gaze shifts between her friend and her teacher, while Rukia glares daggers at her husband's back. It's okay, Rukia. It's not included in the major checklist so- It's unfair, Momo! He's always bossing me around. In school, in the house and everywhere!
Can't he see that I'm doing all of this for issue, him! Momo sigh and law essay competition pats her friend's shoulder, It'll be fine, Rukia. Every thing's gonna be- Why, hey lovely young ladies! A brunette guy appears from hamlet in gertrude essay nowhere, interrupting their conversation. Wanna hang out law essay 2013 india, with a single hot guy like me? the guy asks, placing his arms on both girls' shoulder and pulling them against essays anthem by ayn rand him. The gesture surprises the girls but before they could react, a bag hits the guy square in the face, making him fall hard on law essay competition the ground. Rukia and Momo blinks at the fallen man before turning at coursework the direction where the bag came flying in. Get inside the school grounds, now. Ichigo says as he walks towards the two girls and picks his bag nonchalantly from the ground. The scowl on law essay india his face is deep enough to silence any objections especially from his wife.
Rukia and Momo nod and jog their way towards the school gate while Ichigo stare down at the guy. Uugh. the brunette groans, hands covering his face as he tries to coursework sit up. Oh that hurts, men! What are you, some kind of competition india a- his voice caught in his throat as his eyes meet. Alright, class that would be all, I'll see guys later. Bye. Goodbye, Kurosaki-sensei. the class chorus and starts packing their bentos and in gertrude essay leaves the room. Competition. Chatting, laughing, some are making fun of each other and everything while their teacher goes out ahead of them in a hurry. Rukia's face churns in confusion as she watches her teacher/husband heads out anxiously. Rukia, shall we go now? Momo asks in an enthusiastic manner.
Not removing her eyes at issue research the door, Rukia nods and law essay competition 2013 india grabs her bag. Yeah, let's proceed with the preparation process, she says and the two girls start walking out of the room. Ichigo goes straight out of the school gate and meets with the brunette guy that he encountered earlier. Ichigo! Over here! the brunette calls out and Ichigo walks towards him. Geez, I thought you're not gonna leave that room.
Seriously, how can you stand being a teacher? It's like having a brain cancer. It sucks! Yeah, it sucks. just like us, Ichigo says then is taken aback as the brunette guy hugs him and do you want starts weeping. Oh Ichigo!
I miss you so much, my old friend! It's been many many centuries since we last saw each other! I'm really happy to see my scowling old buddy. Uugh. Keigo let go of me, you- Ichigo groans as he tries to pry his long lost. okay, friend off him, elbowing his face away to escape from the tight hug that the brunette gives him. But Keigo clings tighter and competition so the hamlet in gertrude two starts to wrestle. After 15 minutes. Geez, I can't believe I'm here again! I mean, it's like forever since I last.
Keigo's voice trails off as his gaze wanders around the small secluded shoten house/store/restaurant while Ichigo finds them a place to sit. My, my. A long lost comrade is back in town. 2013 India. It's nice to issue research paper see you again, Asano-kun. Competition India. A man with a bucket hat greets and steps towards them. Ah, it's nice to fences essay see you too, Urahara-san!
Where is Yuroichi-san? She's at the grocery. I'm glad you're able to bring Kurosaki-san here again. It's been awhile since he last ate here. My wife makes a delicious bento so I don't have to go here anymore. Keigo's eyes widen and he rushes towards his friend, You mean, you found your mate already?
And you even marry her? Ichigo crosses his arms and turns his head on law essay 2013 the side, Yeah, I married her 5 years ago. Ooh. how does she look? I mean how does she look? Is she sexy, with large bumps and a roundish- Shut it Keigo! It's for essays anthem by ayn, me to know and for you not to find out!
Keigo whimpers while Urahara chuckles beside him, Kurosaki-san is a bit possessive of his mate so you better find your own mate, Asano-kun. Keigo's face saddens and law essay india he suddenly becomes gloomy, making the other two persons in fences essay, the room look at him in confusion. Oh my, did I say something wrong? Urahara asks. Keigo shakes his head and competition 2013 takes his seat in front of computing Ichigo. He sighs heavily before he starts, Actually, I also found my mate 5 years ago. 2013 India. Her name is Chizuru, she was a tomboy when I first met her but. Luckily, I was able to make her a real woman. Ichigo gives him a dirty look which says 'Of course you will'.
No, Ichigo I didn't rape her or something. It's. well after we had our first sex; she said it's wonderful to be a woman afterall. So that's how she became one. Keigo sighs and looks down his lap. Ichigo can see that his friend is somewhat reminiscing on certain memories in his head so he doesn't speak for a while. His friend, Asano Keigo is also a half blooded vampire. But unlike him, it's Keigo's choice to issue become one. After a moment, Urahara decides to break the law essay competition 2013 india silence. So where is she? She. she left me, Keigo says in essay the taming, a very sad tone and a hurtful look on his face. Ichigo scowls, Why is that?
I don't know. We were happy and we don't have a very serious fight. We're about to get married next year, but as I got in our apartment one afternoon. she's gone. Keigo sighs softly before he continues. Though she left some of her clothes and she even left her glasses at law essay competition india our bedroom.
But it's been two weeks and essay the taming she hasn't come home yet. Is that why you're here in Karakura? Urahara asks. Yeah, I thought I could find her here. And I want to law essay competition india ask for your help too. Anthem By Ayn. Keigo turns at his orange haired friend then. Oh Ichigo, my friend! Please help me find my mate! I beg you! Ichigo groans, Uurgh.
I'll help you but WILL YOU STOP HUGGING ME! A strong punch lands on law essay Keigo's face and he comes flying towards the other side of the room. That would be 2-9-9 for a single chair, Urahara says, grinning widely at the wrecked furniture. Then a crappy painting drops on Keigo's head and breaks into half. Urahara blinks, Oh, I just saw that painting on the street so it'll be for free. Shrew. You don't have to pay for it. Ichigo grimaces at Urahara but the greedy store owner just smile back at him and says, You're welcome. Ichigo goes back to school a bit late that afternoon, thanks to his clingy idiotic friend. That guy knows how to make him all worked up.
But honestly, he missed his friend too. It's been such a very very long time since he last saw a high school friend so it's quite good to see him again. But Keigo's problem really bothers him though. His mate left him, though that was likely to happen since Keigo is competition 2013 a huge pervert. But the hamlet in gertrude essay thing is. She left some of her clothes. And she even left her eye glasses which she always wear, 'cause she has a quite poor eyesight. Ichigo crosses his arms and leans down on his empty classroom wall.
He peers down the window and sees his wife walking towards the field along with her classmates, wearing her PE uniform. Competition 2013 India. She has a big smile on her face as Momo tells her some story with matching hand gestures and all. Then one of her male classmates approaches the two and do you want college asks something. Rukia and Momo smiles and nods at him before jogging on their way to competition india the field. Ichigo's eyes glower. Rukia is the taming shrew still young, she still has a lot to learn and law essay 2013 india experience. What if she fell in love with someone else? What if she decides to research leave him when she finally turns to law essay competition india legal age? His eyes narrow and his hands tighten on the windowsill.
A low growling stirs inside him as his body suddenly tense. Paper. A nerve appears on his jawline as he grits his teeth hard. He closes his eyes and law essay cracks his neck slowly. His feeling hot and anthem by ayn rand there's only one way to cool him down. Rukia, hurry up! We're gonna be late for class! Momo calls, knocking at the door of the cubicle where Rukia is. After their PE class, they go to the shower room to law essay india change back to hamlet essay their school uniform before going to their last class of the afternoon. Go ahead, Momo! I just.
I can't find my spare panty! Then don't change! Just use the 2013 india ones you're already wearing! What? No. By Ayn. It's dirty already. You know me, Momo. I'd rather be naked than wear a dirty underwear. Momo sighs, Alright! Just look at your bag, I'm sure it's right there!
Duh? That's what I'm doing right now. Momo pouts, So, I'll be going ahead! Hurry up there, okay? Okay! Rukia answers and law essay 2013 india hears her friend leaves the room. She sighs and searches for fences essay, her missing undies. Oh men, where is it? she mumbles as she plops down on the bowl, still rambling on her bag, wearing only her bra and PE jogging pants and totally not aware of the law essay figure looming in front of her cubicle door. She grunts in irritation but her underwear are really missing. Looking for something? Rukia jumps from what her seat and her hands cover her front instinctively.
Her eyes wide and competition 2013 india her mouth agapes as she feels like her heart jumps out of her mouth. Geez, Ichigo! You scare the issue research paper hell out of me! she growls and huffs sharply. She grabs her bag to cover more of law essay competition india her body. Want Essay. What are you doing here? This is the girl's shower room, you dope! Rukia scolds but Ichigo's face isn't changing.
And that's when she notices. He's staring intently at her, almost hungrily. His expression tight and serious. his scowl is deep, his eyes stares deeply at hers, he looks tense too if his shallow and ragged breathing is any indication. What's wrong? Is something wrong? she asks out of concern rather than fear of seeing how frightful he looks right now. Law Essay India. She had seen him like that before but he seems more outrageous right now. Rukia's eyes slightly widen as her husband steps inside the small space, not even minding to close the fences essay door behind him. Ichigo?
Rukia. India. Ichigo says lowly as he steps closer to hamlet essay her. Competition 2013 India. Then all of a sudden, he hugs his wife tight against his chest. Rukia. he whimpers again. Confused and startled at his actions, Rukia gently push herself away to look up at him. Ichigo, what are you doing? Someone might catch us here. And the next events happen so fast, Ichigo lifts her up and place her to sit on do you want from essay top of toilet bowl tank. He then settles in competition 2013 india, between her legs, while his hands rest on either side of her face as he buries his face on questions her creamy neck. 'Oh my God.' is all Rukia can think of as her heated husband places small love bites on her neck.
She doesn't know what's gotten into him. Law Essay 2013. First, he barged in the girl's shower room, cornered her inside this cubicle, placed her on computing coursework top of toilet bowl tank and law essay competition india is now feasting on her neck like the night they first met. Rukia takes in a deep breath as she tries to relax against his kisses. Essay The Taming Of The Shrew. The preparation isn't finished yet but if it's her faith to lose her virginity on top of a toilet bowl tank in one of the cubicle inside the school shower room then so BE IT! She puffs out a deep breath as Ichigo's lips traces her collarbone. A shiver runs down her spine and sends a tickling sensation on the tip of toes. Since she's only wearing a bra, there's too much skin for him to offer. Rukia whimpers as she tries to control the bubbly and competition 2013 india tickling sensation that his gentle caress brings her. She and questions the taming of the Momo watched a porn video the 2013 other day as part of The Preparation and their eyes bulged out the entire time watching the act. Of The Shrew. She didn't know that people do that. it's kinda embarrassing for her but Ichigo's kisses and carresses is slowly wiping her fears and embarrasment.
His lips felt wonderful against her skin, she must admit. Though a bit relax, Rukia remains still on her seat as Ichigo moves his lips up to law essay her jaw, ghosting his way to little ear then dragging his lips down to her pert nose. The sensual motion of his lips and hamlet his shallow ragged breathing is like a lullaby making her eyes to droop in pleasure subconsciously. Just relax, he says huskily against her flushed cheek as his hands start wandering on her shoulder and down to her arms. She takes in another deep and tries to do as he says. She can feel her body starts heating up and there's a hot and law essay competition 2013 india bubbly stirring on her lower body as Ichigo lightly licks the corner of her lips then without warning claim her full lips. Rukia stiffens a bit but then relaxes as he starts to move his lips. Do You Want From. His tongue dances gently against her closed lips. As always, there's gentle urgency on his kisses. It's like he's trying to teach her or something so Rukia decides that it's time to learn her lessons and take her part on law essay competition 2013 the kiss. Fences Essay. She closes her eyes and slowly. Competition India. ever so slowly moves her lips apart.
But! She snaps her eyes open as Ichigo suddenly pulls away. You won't leave me, will you? he asks lowly, eyes glowing red and staring intensely at her. With her cheeks burning and her chest slightly heaving, Rukia knits her brows in in gertrude, confusion. What-
You can't leave, Rukia. You are my mate so you'll stay with me forever. Rukia. Ichigo cuts in, resting his forehead against hers and law essay 2013 india trying to coursework control his breathing as his body trembles involuntarily. Flaming eyes stare at her deep purple ones, You can't leave me, okay? Rukia meets his gaze as if searching for something in it before finally nodding. Good. That's good, Ichigo breathes then buries his face on her shoulder to 2013 india calm himself. Essay Questions. Rukia gulps softly as his breath fans her neck. And as they stay like that for a while now, her embarrassment grows again knowing that Ichigo is law essay 2013 india back to his self again. And oh God she guesses right, cause after a moment Ichigo pulls away and questions the taming of the stares at her flushed cheek with that oh so familiar arrogant smirk on law essay his lips.
So, is this the issue one you're looking for? he asks, waving a tiny black satin cloth with small ribbon lace also known as 'panty'. Rukia's eyes widen and her cheeks turn redder, Give me that, you perverted jerk! she reaches for the garment and tries to snatch it but Ichigo holds it up and 2013 india steals a peck on her lips so she fails on getting her underwear from him. Uuggh, she groans as she slumps back on the tank completely forgetting about her half nakedness, pouting and scowling at him. Ichigo chuckles and leans closer to her. Hamlet In Gertrude. Don't worry hun. I'll give it back, but on one condition.
What condition? Rukia asks irritated. And before Ichigo answers her, his eyes roll down at competition her half naked torso then back at her flushed cheeks. He grins. Anthem By Ayn. wickedly. Classes are finally over, and students are now crowding the law essay competition 2013 hallway. Rukia, are you alright? You're walking like a. weird duck. Rukia groans and reaches behind to adjust her underwear before turning at in gertrude essay her friend. Momo, I didn't get to change my underwear. And I feel so gross!
Why? You haven't found the spare? No, I found it. But a dirty rat ate it, she says between gritted teeth that Momo just says a sympathetic, Oh. At the faculty room. Ichigo plops down on his chair and loosens his tie, placing a hand behind his neck and competition india massaging it a bit. You look exhausted today, Kurosaki-kun, Inoue comments, popping out from the taming shrew her cubicle wall and placing a mug of coffee at his table. Ah, no. Actually, I enjoyed the competition india day.
Thanks, 'Especially the little ' foreplay' at the shower room.' That's nice. Inoue beams. Kurosaki. a low cold voice cuts into the scene, making the two orange heads to turn at research paper their side. Oh, Ishida-kun! Inoue greets. Me and law essay competition 2013 Kurosaki-kun are talking about our day. How about you, how's your class today? Their fine.
Thank you for asking, Inoue-san, Ishida Uryuu answers then turns back at his male co-teacher. And I believe that Kurosaki-san enjoyed his day way too much. Ichigo cocks his head and leans further into his chair. Issue Research. Yeah, I really love my job. Ishida huffs lowly and adjusts his glasses before he continues, Of course you do. Competition. But you still have to hamlet in gertrude essay learn some manners. What do you mean? Ichigo asks nonchalantly before seeping on his coffee. In the shower room. There goes his coffee.
Uryuu smirks, I believe you know exactly what I mean now. Ishida, you stalker! Ichigo growls, pointing an accusing finger on the glass wearing teacher. Don't worry, Kurosaki. I'll let it pass this time. Ichigo glares at Uryuu and the guy does the same. Tension rises up around the faculty room as Ichigo and Uryuu stare each other down. Noticing the silent war between the two male, Inoue timidly cuts in the scene. Law Essay India. Ah, excuse me.
Are you- No, the two says in unison. Inoue sweat drops and smiles awkwardly, O-Okay. I'm going now, Uryuu says. Over Rand. See you on Monday, Inoue-san. Ah, see you on Monday Ishida-kun. Take care! Geez, that Ishida knows how to ruin my day, Ichigo says making Inoue to turn back at him. Oh, Kurosaki-kun. You still have something on your chin.
Ah. Ichigo reaches up and tries wiping it. Wait, I have some tissue in my bag. Let me just get it. No, thanks. I have my handkerchief here. Law Essay Competition 2013 India. Ichigo reaches for a piece of paper cloth in his pocket and wipes it on his mouth.
Rukia and Momo happen to 2013 india pass by the faculty room where they see the two teachers. They watch the two for awhile. Inoue comments something about his face and Ichigo wipes his face immediately. Inoue then searches for her bag while Ichigo takes out hamlet in gertrude, something from his pocket and uses it to law essay 2013 wipe dirt off his mouth. But it takes a split second for Rukia to notice the thing that he's using to fences essay wipe his mouth. It's not a handkerchief but an competition underwear! Her underwear! Her eyes widen 'cause Ichigo isn't aware of what want what he's using and law essay competition 2013 Inoue-sensei is anthem by ayn rand turning back at him. Oh, found it! Here-
Every teacher in law essay competition india, the faculty room turns at the door after a petite student suddenly barge in. No yelling at fences essay the corridor especially inside the faculty room! an old female teacher scolds, yelling herself making everyone to law essay india sweat drop. I'm sorry, Megumi-sensei. Rukia apologizes using her high pitch tone. Over Anthem. I just need to. Law Essay Competition. tell someting to do you want from college Inoue-sensei. The auburn haired teacher looks at her quizically. Rukia succeeded on stopping her from turning on her husband and seeing that embarrassing event. Oh, what is competition 2013 it Kuchiki-san? the teacher smiles. I just. Of The Shrew. want to tell you. 2013 India. that. she says, pausing every now and then to glare at hamlet her husband and the offending thing on his hand. But Ichigo just raises his eyebrows and blinks at her. That. ahm. you. are nice, she finishes.
Inoue smiles widely at her, Oh that's so sweet of you. I knew you were. Law Essay 2013 India. the auburn teacher trails off but Rukia didn't pay attention at what she's saying cause she's too busy sending glaring messages to her husband. Momo is also giving him signals and warning like she is fences essay playing a charade or something. Ichigo stares at the two girls and when he finally realizes what they want to say, he turns at the thing on his hand. and grins. Rukia's jaw drops watching as her husband plays with her underwear and twirls it around using his index finger instead of hiding it back on his pocket. . and india we teachers really appreciate that. Right, . Hamlet In Gertrude. Kurosaki-kun? Inoue finishes, turning and competition 2013 india smiling at the orange haired teacher who's sitting casually on want essay his chair with both hands now inside his pocket. Yeah, that's right, he says nonchalantly, glancing at his gaping wife with a devious smirk. Great!
She forgets that he's a fast moving monster. 'Uugh! That jerk!' Rukia groans to law essay 2013 herself then sighs in relief. So, is there anything else you want to say, Kuchiki-san? Inoue asks, taking a seat next to Ichigo. No, that would be all. Rukia forces a wide smiles then tugs at her still dumbstruck friend. C'mon, Momo let's get outta here. Bye Kuchiki-san, Hinamori-san!
I'll see you next- NO YELLING INSIDE THE FACULTY ROOM! After spending one hour inside her bathroom, Rukia finally emerges wearing a short purple silken robe and with a white towel around her head. Her eyes slowly open as she tilts her chin up before walking inside her bedroom. This will be the issue research paper night of law essay competition all nights. Fences Essay. It's the law essay india night that they're waiting for.
The fateful night of pure love and romance. The night that she will surrender her body and fences essay soul. The Preparation had all been done. the balloons had been sent to 2013 india the heavens, the shrew full moon is shining brightly, the candles are lit, the bed is 2013 india perfect, she is perfect, even the pain killers are set perfectly, everything's perfect and ready. except one. Where's that idiot? Rukia groans as she glances at the clock then pace and back inside her room. It's already 9 in the evening but her husband is anthem anywhere but their house. She texted him but he's not replying yet. Speaking of law essay competition india replying, Rukia grabs her phone and quickly opens her new message. 'B ryt der in 5 mins.'
Rukia bites her lips to suppress her smile then types in her reply. 'Ok. ^-^' She tosses her phone aside and lets out a suppress squeal. She didn't realize that this night will come that she will become one with Ichigo and that she'll be so excited about want essay it. Rukia stands in front of law essay 2013 her full length mirror and removes the fences essay towel on her head. She combs her raven hair softly then applies some lotion on her skin. She starts untying the knot of her robe before letting it fall down on the floor before staring back at her image in the mirror. She's wearing a silky white spaghetti strap lingerie that falls down to law essay 2013 her mid thigh exposing the creamy delicate skin of her legs. It has purple flowers embroidered along its v-shaped neckline which makes it look more sexy and appealing. Now where did she get that?
It's my Mom's lingerie. She hasn't worn it yet so it's still new. C'mon, it'll fit perfectly on you.' Yeah, Momo is the best. Rukia smiles at her image, she's petite but her curves are developing nicely. Computing. She grins and starts walking slowly towards the mirror. I may not have the big chest like Inoue-sensei, she says swaying her hips to side. I may not have the curves of a model, she then moves her hands to her waist and drags it slowly up to her head to 2013 mess up her hair as she starts doing a slow sexy dance in front of her mirror.
I may not have the longest leg to die for what, but I. am still. the most gorgeous girl. in the whole wide-waaah! she yelps and law essay india jumps on essay shrew her feet as she sees the image of competition india a man standing on her door. Ichigo. What are you doing? he asks, walking towards his beat red wife. All of her confidence vanishes through the window and she quickly grabs her blanket to cover her body. all shy and blushing furiously. Ichigo, I-I'm just. I. Rukia stutters as Ichigo leans down and pecks her on the cheek. Rukia, I'll be out for the night, is all he says before he walks out of her room casually as if he didn't witness anything. What?
Rukia exclaims, draping her blanket around her body before following him at coursework his room. I said, I'm going out tonight. I need to help my friend find someone, he says, grabbing a black hooded jacket and starts changing his clothes. But you can't go out tonight. Rukia, this is serious. I'm the competition only one who can help him so I have to go. But, but what about the full moon, and the candles, the hamlet essay bed and the pain killers? Rukia blushes, I-I mean why not go tomorrow.
It's easier to find someone during daytime. For humans, yes. But we're vampires, we're more functional at night. My friend's mate who left him 2 weeks ago without a word, Ichigo trails off as he pulls on 2013 his jacket. So we're trying to trace her here in Karakura since this is his mate's hometown.
What? he asks smirking down at the taming shrew her disappointed expression. Law Essay. Rukia looks up at him and is a bit distracted at how he looks. Seriously, without the dress shirt and tie, Ichigo looks like a tall teenager. very tall teenager. Not receiving an answer from research paper his blushing wife, Ichigo slowly steps closer to her. You're gonna miss me, huh? Rukia snaps from her stupor and scowls. No, I'm won't, she pouts and turns away from him. Ichigo smirks and in competition, one swift move, he's able to rid of the blanket that's covering her. Rukia gasps in surprise then she feels his arms around her waist and pulls her up against him. Her feet touch nothing but air as Ichigo holds her body against him so that their eyes are level with each other. I like your dress.
You look beautiful and sexy, he says bluntly, with a small smile playing on the corner of his lips. With another deep blush, Rukia turns her gaze down at her hands press up against his chest and mutters, Thanks. Ichigo smiles and lean towards her to capture her lips. Rukia doesn't fight back. She just closes her eyes and enjoys that sweet kiss, making slow and shy movements with her lips. Ichigo notices her response and it surprises him a bit. But as much as he wants to go further with his wife, he has to go.
He has to stop. And what better way to make them both stop than to. Aah! Rukia squeals as she feels him squeezing her ass' cheek hard. Ichigo! she whines and pulls away from his arms, blushing furiously. Ichigo laughs at her cute reaction and lets her go. He then turns at the door and paper opens it. I'll be going now, baby. Be sure to lock your door, cause I might come crawling at competition india you, he says, winking at her before closing the paper front door.
Hmp! Pervert. Rukia pouts and stares at law essay 2013 india the closed door before sighing and walking at her room, disappointed. She plops down on her bed then composes a message on her phone. 'Momo. I really think we should've bought the white balloons .' Her name's Patrice, a college student and computing coursework a half blood, Abarai Renji says, staring at the lifeless body of a girl they find lying at the dark alley. Her internal appendages and competition india bladders were torn out from the large whole on her stomach, and a lot of bitten flesh all over essays over anthem rand her body. The sight is sickening but he and the short guy beside him couldn't care less of how gruesome the scene is. They are more concern for who causes her death. Found any clues, Toshiro?
Renji asks. The silver haired guy kneels down on one knee and brushes a blood from the girl's wound. He sniffs on competition it and tries to trace something but then he shakes his head. Negative. There's no smell on it, just pure blood. Even the hamlet in gertrude bite marks and scratches are hard to law essay competition 2013 india recognize. No smell, no pattern, no sign, no clue or whatsoever! Damn! Who's behind all of this? Renji spats, punching the anthem rand wall angrily. Calm down, Abarai.
Toshiro says, standing up from the ground. Two more days and we're heading back to Japan. I think this will do it. What do you mean? Toshiro places one hand inside his coat's pocket and looks at Renji in sideways before saying, We have to reunite and rebuild the organization. Yes, we will gather the members of the Vampire society. Hiding in the shadows of the competition 2013 trees a few meters away, a pair of glowing eyes narrow at the two. Watching and listening intently at want from college essay their conversation. And upon hearing the news about the society, a wide wicked grin curves on 2013 india his lips. Everything is hamlet essay starts to flow with the plan. So now, he has to leave and law essay 2013 send the good news to do you want essay the leader of their clan.
Add me up on Facebook okay! And oh, DON'T FORGET TO REVIEW! OR ELSE ICHIGO WILL STEAL YOUR UNDERWEAR. HEHE, IS IT A GOOD THING OR A BAD ONE? ^.