Bacterial Artificial Chromosomes (BACs) Features

Bacterial Artificial Chromosomes (BACs) Features Definition: Bacterial Artificial Chromosomes or BACs are plasmids (circular DNA molecules) constructed with the replication origin of E.coli F’ Factor. F’ is an incompatibility group involved in E. coli conjugative ability and chromosomal transfer, which can exist as an extra-chromosomal element. 1st developed as a large insert cloning system to facilitate the construction of DNA libraries to analyze genomic structure. Technology was developed to carry out genetic and functional studies of viruses (herpes virus especially). Since then BACs application have grown intensely and have benefited the research community in many fields, such as in genomic fingerprinting, sequencing of the human genome, in vaccine development and in vitro transgenesis,. Characteristic features of BAC vectors The original BAC vector, pBAC108L, is based on a mini-F plasmid, pMBO131 (Figure 1) which encodes genes essential for self-repli ­cation and regulates its copy number inside a cell. The unidirectional self-replicating genes are oriS and repE while parA and parB maintain copy number to one or two for each E. coli genome. Multiple cloning sites is present, flanked by â€Å"universal promot ­ers† T7 and SP6, all flanked by GC-rich restriction enzyme sites for insert excision.   Presence of cosN and loxP sites(cloned in by bacteriophage l terminase and P1 Cre recombinase, respectively) permits linearization of the plasmid for convenient restriction mapping. There is a chloramphenicol resistance gene for negative selection of non-transformed bacteria. Vector is 6900 bp in length and is capable of maintaining insert DNA in excess of 300 kilobases (kb). Other BAC Vectors There have been many modifications done to increase the ease-of-use as well as for use in specific systems and situations.   pBeloBAC11 2 and pBACe3.6 are modified BAC vectors based on pBAC108L and are commonly used as a basis for further modification. pBeloBAC11 The primary characteristic of this vector is the addition of a lacZ gene into the multiple cloning site 2 of pBAC108L. Plates supplemented with X-gal/IPTG, an intact lacZ gene encodes b-galactosidase which catalyses the supplemented substrate into a blue substance. Successful ligation of insert DNA into the vector inactivates lacZ, generating white colonies, indicating the presence of a successful vector-insert ligation. It is still a low-copy number plasmid due to presence of parA and parB. Size of vector is 7507 bp in length. pBACe3.6 This vector is based on pBAC108L but is more highly modified than pBeloBAC11. In order to overcome the issue of low plasmid copy numbers, the P1 replicon in F’ was deleted and a removable high copy number replicon originating from an inserted pUC19 was introduced.   This vector contains 2.7 kb pUClink stuffer fragment which is flanked by two sets of six restriction sites within a sacB region. Levansucrase, a product of sacB gene, which converts sucrose (sup ­plemented in the media) to levan, which is toxic to E. coli host cells. Hence, if the vector is re-ligated without an insert, the functional sacB produces levansucrase and the cells die before forming colonies. Successful ligation of an insert into the vector increases the dis ­tance from the promoter to the coding region of sacB, disrupting toxic gene expression in the presence of sucrose. In addition to this vectors, there are many specialized BAC vectors carrying a variety of different combinations of drug resistance genes. Besides, many different selection mechanisms and markers are available. Modifications of cloning sites (unique restriction endonuclease sites) are also common as per the addition of genes and promoters specific to different strains of bacteria. Development of BAC vector Advantages of BAC Vectors The large size of BACs help to minimize site of integration effects, a phenomenon which has been defined as endogenous sequences (such as gene coding regions and distal regulatory elements) to be disrupted, and to produce potentially undesirable phenotypes in gene cloning technology. Endogenous gene expression more accurately than other cloning systems. The human genome BACs consist of the full gene structure(which play very important role in gene regulation). Therefore the human genome BACs will ensure full mRNA processing and splicing when genes are transcribed, and produce the full complement of protein isoforms once mRNAs are translated. It can be transfected and expressed in mammalian cell lines even if transfection efficiency and copy numbers are low. Disadvantages of BAC vectors A construct containing a large genomic fragment is likely to contain non-related genes which may lead to indirect, non-specific gene expression and unanticipated changes in the cell phenotype. Recombinant BAC constructs can be time-consuming and labor-intensive. The large size BAC DNA constructs are more easily degraded and sheard during manipulation before transfection. Applications of BAC vectors BACs are useful for the construction of genomic libraries but their range of use is vast. It spans from basic science to economically rewarding industrial research, and fields as prosaic as animal husbandry. In genomic analyses, it helps in determining phylogenetic lineage det between species. Helps in study of horizontal gene transfer and since bacterial genes are usually clustered, the ability of BAC vectors to accommodate large inserts has allowed the study of entire bacterial pathways. By isolating DNA directly from soil or from marine environments, the â€Å"metagenomes† of those organisms which are either uncultureable or are termed viable but uncultureable can be cloned into BAC vectors and indirectly studied. In industrial research fields where BAC vectors are invaluable tools in cataloguing novel genomes is in the discovery of novel enzymes. Work has been done on identifying enzymes that are involved in biopolymer hydrolysis or even radioactive waste management. BAC vectors have been instrumen ­tal in studying large double stranded DNA viruses both from an academic point of view and as a tool to develop improved vaccines. In genomic research, high throughput determination of gains and losses of genetic material using high resolution BAC arrays and comparative genomic hybridization (CGH) have been developed into the new tools for translational research in solid tumors and neurodegenerative disorders. BAC technology is becoming the most upcoming method for genome sequencing. The technique uses an overlapping tailing part of large genomic fragments (150-200 kb) maintained within BACs. Every individual BAC is shotgun sequenced, where these large overlapping sequences of the BACs are assembled to produce the whole genome sequence. BACs have also been used in mammalian genome mapping, genomic imprinting, vaccine development, gene therapy and studies of the evolutionary history and functional dynamics of sex chromosomes have recently been possible using BAC libraries. YAC (yeast artificial chromosome) vectors Definition: Yeast artificial chromosomes (YACs) are plasmid shuttle vectors capable of replicating and being selected in common bacterial hosts such as Escherichia coli, as well as in the budding yeast Saccharomyces cerevisiae. They are of relatively small size (approximately 12 kb) and of circular form when they are amplified or manipulated in E. coli, but are rendered linear and of very large size(several hundreds of kilobases), when introduced as cloning vectors in yeast. Many different yeast artificial chromosomes exist as ongoing refinements of the initial pYAC3 and pYAC4 plasmids (Figure 1) constructed by Burke et al. (1987). Basic structural features of YACs were developed from the yeast centromere shuttle-plasmids (YCp) series. These are composed of double-stranded circular DNA sequences carrying the b-lactamase gene (bla) and the bacterial pMB1 origin of replication, thus conferring resistance to ampicillin and the ability to replicate in bacteria, respectively. YACs also contain the cloning site in the middle of the SUP4 suppressor of an ochre allele of a tyrosine transfer RNA gene; this enables restoration of the normal white color phenotype in otherwise red ade1 and/or ade2 nonsense mutants. Accordingly, in the insertional inactivation cloning process, the SUP4 gene is disrupted by the DNA insert, thus removing the suppression of the ade mutations and allowing their phenotypic expression as red color. They also include yeast ARS1 with its associated CEN4 DNAsequence, as well as the URA3 selectable marker. Biological Features of YACs The stability of YAC vectors in yeast per se is similar to that of natural chromosomes provided that all three structural elements (ARS, CEN and TEL) are present and functional, in addition, that the minimal required size is reached by the insertion of enough exogenous DNA. Indeed, several mutations are known to affect YAC stability and segregation together with natural chromosomes. Another important consideration is that faithful duplication of YACs is guaranteed only if other DNA sequences incompatible with ARS do not exist on the construct, particularly relevant when unknown DNA inserts are cloned in the YAC vector, as in the case for genomic libraries, in which there could be cryptic or otherwise unknown ARS-like sequences able to interfere with the ARS function. Construction of YACs Steps: Initially, purification of plasmid DNA is carried out. Two distinct digestions are performed: the first with BamHI that cuts twice adjacent to the two telomeric DNA sequences flanking the HIS3 gene, which therefore is excised from the plasmid and lost (Figure 2a). This first digestion generates a long linear fragment carrying telomeric sequences at each end. The second digestion consists of the opening of the cloning site within the SUP4 gene (Figure 2a). As a result of this second digestion, two linear fragments are produced as left and right arms of the future linear YAC (Figure 2b). Large DNA fragments with ends compatible to the cloning site, obtained from the desired genome source by digestion with an appropriate restriction endonuclease, are ligated with phosphatase treated YAC arms, to create a single yeast-transforming DNA molecule (Figure 2c). Primary transformants can be selected for complementation of the ura3 mutation in the host, and successively for complementation of the host trp1 mutation, thereby ensuring thepresence of both chromosomal arms. Transformant colonies containing the exogenous DNA insert within the SUP4 gene are detected by their red colour, due to the inactivation of the suppressor activity and the consequent accumulation of a red metabolic precursor in ade host cells. Applications of YACs Applications of YACs range from generating whole DNA libraries of the genomes of higher organisms to identifying essential mammalian chromosomal sequences necessary for the future construction of specialized mammalian artificial chromosomes (MACs). Helps in the study of regulation of gene expression by cis-acting, controlling DNA elements, that are present either upstream or downstream of large eukaryotic genes, after the transfer of these YACs from yeast to mammalian cells. YAC libraries has greatly advanced the analysis of genomes previously cloned in cosmid vectors. For example, YAC clones have been used as hybridization probes for the screening of cDNA libraries, thus simplifying the characterization of unidentified genes. Recent technological developments allow the transfer of YACs into mouse embryonal stem (ES) cells and the subsequent generation of transgenic mice. Investigators have begun to employ these artificial chromosomes for the in vivo study of multigenic loci in mammalian cells. Two process can be used to obtain a sequenced genome, or region of interest: 1. Physical Mapping. 2. Chromosome Walking. It allows for the detailed mapping of specific regions of the genome. With the help of this, whole human chromosomes have been examined, such as the X chromosome,generating the location of genetic markers for numerous genetic disorders and traits. Bibliography Smith, GA. Enquist, LW. 1999 A self-recombining bacterial artificial chromosome and its application for analysis of herpesvirus pathogenesis. Proc. Natl. Acad. Sci. 97; 4873-4878 Shizuya, H., Birren, B., Kim, UJ., Valeria, M., Slepak, T., Tachiiri, Y., Simon, M. 1992 Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. Proc. Natl. Acad. Sci. 89; 879-8797 Fu, H., Dooner, HK. 2000 A gene-enriched BAC library for cloning large allele-specific fragments from Maize: Isolation of a 240-kb contig of the bronze region. Genome Res. 10; 866-873 Kim, UJ., Birren, BW., Slepak, T., Mancino, V., Boysen, C., Kang, HL., Simon, MI., Shizuya, H. 1996 Construction and characterization of a human bacterial artificial chromosome library. Genomics 34;213-218 Frengen, E., Weichenhan, D., Zhao, B., Osoegawa, K., van Geel, M., Jong, PJ. 1999 A modular, positive selection bacterial artificial chromosome vector with multiple cloning sites. Genomics 58; 250-253 Flotte, TR. 2000 Size does matter: overcoming the adeno-associated virus packaging limit. Respir. Res. 1; 16-18 Whitman, WB., Coleman, DC., Wiebe, WJ. 1998 Prokaryotes: the unseen majority. Proc. Natl. Acad. Sci. 95; 6578-6583 Anderson, SI., Lopez-Corrales, NL., Gorick, B., Archibald, AL. 2000 A large-fragment porcine genomic library resource as a BAC vector. Mamm. Genome 11; 811-814 Heintz, N. 2001 BAC to the future: The use of BAC transgenic mice for neuroscience research. Nature Rev. Neur. 2; 861-870 Adler, H., Messerle, M., Koszinowski, UH. 2001 Virus reconstituted from infectious bacterial artificial chromosome (BAC)-cloned murine gammaerpesvirus 68 acquires wild-type properties in vivo only after excision of BAC vector sequences. J. Vir. 75; 5692-5696 Fischer CR (1969) Enzymology of the pigmented adenine requiring mutants of Saccharomyces cerevisiae and Schizosaccharomyces. Biochemical Biophysical Research Communication 34: 306–310. Cross SH, Allshire RC, McKay SJ, McGill NI and Cooke HJ (1989) Cloning of human telomeres by complementation in yeast. Nature 338:771–774. Jakobovits A, Moore AL, Green LL et al. (1993) Germ-line transmission and expression of a human-derived yeast artificial chromosome. Nature 362: 255–258.

René Descartes Essay

Philosophy begins as an art of wondering. This art of wondering, leads man into asking a number of questions about himself and the world around him. Variations in philosophical thoughts as result questions posed by man, help in modification of philosophy. The purpose of this essay therefore, is to do a comparative analysis of Sophie’s World by Josteen Gaarder with three philosophical texts: George Berkeley’s Treatise on Human Knowledge, John Locke’s Essay on Human Understanding and Rene Descartes’ Passions of the Soul. We shall also show the roles of authors and texts in Philosophy. TREATISE ON HUMAN KNOWLEDGE VERSUS SOPHIE’S WORLD One of the outstanding efforts of Berkeley in this work was to show that the world exists as it does because of the ideas perceived by the mind of God and as such, existence of things depends on their perception through the senses. Likewise in Sophie’s World, Aristotle through his belief showed a similarity with Berkeley’s stance in this text Treatise of Human Understanding. For Aristotle, highest degree of reality is what we perceive with our senses. Berkeley also said that existence is the state of being perceived by a perceiver, but on the contrary, Descartes in Sophie’s World believed that he exists because he thinks. It furthermore means that his existence depends on his ability to think. He used such words like cogito ergo sum (latin) and je pense, donc je suis (French) all meaning `I think therefore I am` to illuminate his thesis on this. Plato while stressing the sovereignty of reason in Sophie’s World said that the reason can only give us the true reality of things and as such contradicted the empiricist approach of Berkeley. ESSAY ON HUMAN UNDERSTANDING VERSUS SOPHIE’S WORLD John Locke illuminated in this work that idea is derived from experience of sensation or reflection, just like it reflected in Sophie’s World in which his fellow empiricists likewise stressed the inevitability of experience as the source of true knowledge. David Hume in particular established that man has two types of perceptions, namely: impressions and ideas. By `impressions` he means the external sensations of reality. By `ideas` he means the recollections of such impressions. Aristotle the trail blazer of empiricism, in alignment with the empiricists believed our reason to be completely empty until we have sensed something. John Locke in this book made an attempt to refute the notion of `innate ideas` by proving that it does not exist, whereas in Sophie’s World, philosopher like Plato believed in an innate idea, an external world of ideas outside of the natural world. Descartes even believed that the idea of God is innate. PASSIONS OF THE SOUL VERSUS SOPHIE’S WORLD Descartes defined passions of the soul as a mental state or thought that arises as a direct result of brain activity and such passions can move us to action. He also suggested that one needs to learn to control one’s passions, for they can move one to perform vicious acts. Benedict Spinosa in Sophie’s World, suggested that we must free ourselves from our feelings and passions only then shall we find happiness. Conversely, also in Sophie’s World, the Epicureans through the teachings of Aristippus believed that the aim of life is to attain the highest possible pleasure or passions and by so doing, they contradict Descartes belief. AN EYE ON THE ROLES OF AUTHORS AND TEXTS IN PHILOSOPHY Authors in philosophy play incredible roles in modification of philosophy through their texts. There various attempts to answer the underlying questions of nature and man have led to the developments of dialectical methods like thesis, antithesis and synthesis used in philosophy today. An eye on the three authors and philosophers: Descartes, Berkeley and Locke whose works where compared with Sophie’s World above showed the vital role they played in philosophy. While Descartes was a rationalist, Berkeley and Locke were empiricists, but through their texts, they contributed in `epistemology` or theory of knowledge. CONCLUSION Having done these comparisons, we can see the successful deduction of similarities and differences between the Sophie’s World and three aforementioned philosophical texts. I attest that Sophie’s World is necessary philosophical paraphernalia needed by a neophyte in Philosophy as seen from its versatility in the course of this comparison done, and also how it covered aspects of history of Philosophy in such a sublime manner. POPE JOHN PAUL II MAJOR SEMINARY OKPUNO, AWKA NAME: UMENABA BENJAMIN C COURSE: INTRODUCTION TO PHILOSOPHY COURSE CODE: PHIL 101 REG NO: PJPS/NAU/013/O69 LECTURER: REV. FR DR. AUGUSTINE OBUROTA TOPIC: A COMPARATIVE ANALYSIS OF SOPHIE’S WORLD WITH THREE PHILOSOPHY TEXTS WITH AN EYE ON ROLES OF AUTHORS AND TEXTS IN PHILOSOPHY DATE: 28TH NOVEMBER, 2012 OUTLINES INTRODUCTION TREATISE ON HUMAN KNOWLEDGE VERSUS SOPHIE’S WORLD ESSAY ON HUMAN UNDERSTANDING VERSUS SOPHIE’S WORLD PASSIONS OF THE SOUL VERSUS SOPHIE’S WORLD AN EYE ON THE ROLES OF AUTHORS AND TEXTS IN PHILOSOPHY CONCLUSION.

The Effects Of A Nation s Industrialized Food System Essay

The Effects of a Nation’s Industrialized Food System Our nation’s modern industrial farming has become more than only feeding people; it has become a way for the food industry to make more money as human population continues to grow. The food industry has transformed not only how people eat, but also has had negative effect on our climate as a result of factory farming as illustrated by Anna Lappe in â€Å"The Climate Crisis at the End of Our Fork†. Jonathan Safran Foer in his book Eating Animals, illustrates the effects factory farming has had on animals meant for human consumption. Furthermore, Foer asks many questions to the reader on what will it take for us to change our ways before we say enough is enough. The questions individuals need to be asking themselves are: how do we deal with the problem of factory farming, and what can people do to help solve these issues? Namit Arora in the article On Eating Animals, as well as Michael Pollan in his book The Omnivore’s Dilemma, both address some of the issues that animals face once they hit the kill floor. Eric Schlosser in Fast Food Nation, also writes on the topic of animal abuse, and what goes unseen within the food industry as well as Bernard Rollin and Robert Desch in their article Farm Factories, both demonstrate what is wrong today with factory farming. The food industry today has impacted all facets of farming practices: from our Earth’s climate to the abuse animals endure, and the loss of old world agricultural farmingShow MoreRelatedInflammatory Bowel Disease and Industrialization Essay1682 Words   |  7 Pagesconsidered a fatal condition, painful and disabling symptoms can have a profound detrimental effect on patients’ quality of life. Current understandings behind the etiology of IBD emphasize genetic predispositions to gastrointestinal immune system imbalances. 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The Great Gatsby And The American Dream Essay - 1073 Words

The psychological concept of Abraham Maslow’s Hierarchy of Needs is a pyramid of desirable needs organized based on the fundamentality of the stage emphasizing how â€Å"some needs take precedent over others† (Boeree). One should only move up in levels through the pyramid of needs once he has achieved the needs below. Maslow’s Hierarchy is ordered through the needs of physiological, safety, belonging, esteem and self-actualization. Often times, when individuals are preoccupied with achieving a goal of self-actualization, or maximizing their full potential in life, a few stages are skipped. This individual is not truly self-actualized. In The Great Gatsby and Death of a Salesman, the idea of the American Dream is presented differently; through†¦show more content†¦Similarly, Willy distances himself from his family due to his depression that occurs because he cannot achieve his American Dream. Willy commits suicide in the end to give his sons a chance to fulfill his American Dream of financial success for him because Willy felt he was incapable. In trying to maximize his full potential, Willy lets his family down by hurting his sons’ self esteem and devastating his wife with his death. Willy never became self-actualized himself but wanted his children to continue his legacy. Both Gatsby and Willy chased after their Dream but were unable to catch it. Willy reached for financial success and social status but sacrificed love while Gatsby attained financial success, compromising his character, to reach for love. In the end, they both lose the most basic need on the hierarchy - physical health. Gatsby is murdered because he sacrifices literally everything for Daisy’s love and Willy takes his own life, frustrated because he was unable to achieve his American Dream. Both appear to die selflessly, Gatsby sacrificing himself to save Daisy and Willy committing suicide to give his children an opportunity to thrive; but in reality , their shared infatuation of the American Dream prompts them to give up their lives because they know that they will never fulfill their lives’ potential and become self-actualized. F. Scott Fitzgerald and Arthur Miller both critique the corruption of the American Dream as theShow MoreRelated The Great Gatsby - The American Dream Essay767 Words   |  4 Pagesmade by Marius Bewley’s critical essay â€Å"Scott Fitzgerald: The Apprentice Fiction†, â€Å"Fitzgerald’s ultimate subject is the character of the American Dream in which, in their respective ways, his principle heroes are all trapped.†, can be justified through Scott Fitzgerald’s novel The Great Gatsby and his short story â€Å"Winter Dreams†. 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