ANNOTATED BIBLIOGRAPHY

ATSDR - Cancer and the Environment - How Have Cancer Trends Changed Over The Past Few Years." ATSDR Home. US Department of Health and Human Services, 1 Apr. 2010. Web. 10 Jan. 2011. <http://www.atsdr.cdc.gov/risk/cancer/cancer-trends.html>.

Online webpage. Malignant neoplasm, the uncontrolled growth of abnormal cells in the body more commonly known as cancer, has become the focus of modern biological research. Cancer was first hypothesized and observed by Hypocrites in ancient Greece. From 1975 to 1990, the number of cancer deaths in the United States skyrocketed before stabilizing in 1994. While the number of cancer deaths decreased by 1.4% per year from 1994 to 1998, cancer death rates have once again stabilized. Researchers have been working for years to find methods and procedures to prevent and cure the disease. Most of this research has been focused on cancer gene discovery, the process of finding and mapping oncogenes in human chromosomes responsible for cancer proliferation, as a prerequisite for medical solutions. The discovery of TSGs is a major focus as well, accompanied by functionally analyses of identified TSGs.

Bagchi, A., C. Papazoglu, Y. Wu, D. Capurso, M. Brodt, D. Francis, M. Bredel, H. Vogel, and A. A. Mills. "CHD5 Is A Tumor Suppressor At Human 1p36." PubMed Central - Genome Biology. U.S. National Library of Medicine, National Institutes of Health, 9 Feb. 2007. Web. 10 Jan. 2011. <http://www.ncbi.nlm.nih.gov/pubmed/17289567>

Lab report. Researchers under Professor Anindya Bagchi from the University of Minnesota completed in 1977 in which genomic analyses of developed cancerous cells revealed consistent deletions in 1p36, the outermost band of the short arm of human chromosome 1. The missing 1p36 genetic sequence remained unknown for 30 years. Researchers in 2007 repeated the 1977 study and used advanced sequencing technology to pinpoint chromodomain helicase DNA-binding protein 5 (CHD5). Conducting a functional study, researchers demonstrated how CHD5 functioned as a tumor suppressor in vivo which controlled proliferation, apoptosis, and senescence of cancer via the p19(Arf)/p53 pathway.CHD5 is a member of proteins known as chromatin remodelers which are actively involved in DNA damage repair in human cell systems.

Carlson, Corey M., Joel L. Frandsen, Nicole Kirchhof, R. Scott McIvor, and David A.Largaespada. "Somatic Integration of an Oncogene-harboring Sleeping Beauty Transposon Models Liver Tumor Development in the Mouse." Proceedings of the National Academy of Sciences. The National Academy of Sciences of the United States of America, 07 Oct. 2005. Web. 26 Aug. 2010. <http://www.pnas.org/content/102/47/17059.abstract>.

Lab report. Researchers from the University of Minnesota observed how well the SB transposon system facilitated integration of an activated NRAS oncogenes in mouse hepatocytes to induce tumor growth. Transgenic overexpression of an oncogene induces tumor growth in mice. However, the continuous expression of germline transgenes in somatic cells does not emulate the spontaneous activation of a protooncogene by mutation in an adult somatic cell. The system requires the production of unique transgenic animals for each gene of interest. While viral vectors have been used, research has shown low utility for the delivery of genetic elements to nondividing cells. SB, a synthetic transposon system resurrected by mutagenesis of inactive salmonid transposable element, has been successfully used as a nonviral method to integrate therapeutic genes in the lung, liver, and brain of the mouse. In this study, p19Arf-null and heterozygous recipients developed multifocal liver cancer. The resulting tumors were carcinomas of hepatocellular and biliary origin.

Chou, Danny M., Britt Adamson, Noah E. Dephoure, Xu Tan, Amanda C. Nottke, Kristen E. Hurov, Steven P. Gygi, Monica P. Colalacovo, and Stephen J. Elledge. "A Chromatin Localization Screen Reveals Poly (ADP Ribose)-regulated Recruitment of the Repressive Polycomb and NuRD Complexes To Sites of DNA Damage." PNAS 107.43 (2010): 18475 8480. PubMed. U.S. National Library of Medicine, National Institutes of Health, 30 Aug. 2010. Web. 10 Dec. 2010. <http://www.ncbi.nlm.nih.gov/pubmed>.

Lab report. Poly (ADP ribose)polymerase (PARP-1), an enzyme associated with niacin oxidation-reduction reactions, has been shown to play a role in DNA damage repair. PARP-1 is appealing for researchers working with chromodomain helicase DNA-binding protein 5 (CHD5) because of the enzyme’s structure and activity. This study, studying PARP-1 interactions in Drosophila, observed the role played by PARP-1 in inducing a localized relaxation of chromatin structure allowing DNA repair complexes to form and accomplish repair. Relaxation is achieved through PARP-1’s two zinc fingers which allow the enzyme to bind to DNA at single breaks in DNA strands. When PARP-1 binds to DNA, a collection of covalent and noncovalent interactions between negatively charged histones and PARP-1 unravel genetic information. As DNA damage in repaired, an enzyme glycohydrolase will degrade the PARP-1 allowing the coil to be restored.

Cooper, Tim F., and Jack A. Heinemann. "Postsegregational Killing Does Not Increase Plasmid Stability but Acts to Mediate The Exclusion of Competing Plasmids." Thesis. Ed. Joshua Lederberg. Department of Plant and Microbial Sciences, University of Canterbury, New Zealand, 2000. PNAS. Proceedings of the National Academy of Sciences, 23 Feb. 2000. Web. 29 June 2010. <http://www.pnas.org/content/97/23/12643.full.pdf>.

Lab report. In 2000, New Zealand’s Department of Plant and Microbial Sciences formed a new hypothesis for the evolution of PSK systems in bacteria. Contrary to the view that PSK systems exist to help plasmids stabilize in bacterial populations, they exist only to reduce plasmid competition in accordance with the study. Experimental results were consistent, showing PSK systems as being only selected in environments where plasmids needed to compete with others to achieve horizontal reproduction across bacterial species. By this new “competition” hypothesis, success of PSK systems is consequential of plasmid to plasmid competition by environmental limitations instead of the adaptive nature of plasmids in relation to the bacterial host. Plasmids have even been observed as to go as far as sacrificing several hosts and even themselves to maintain a competitor-free environment. Much research remains concerning the seemingly harmful process of PSK.

"Discovery Genomics, Inc. - Gene Therapy: Sleeping Beauty Transposon System." Discovery Genomics, Inc. Gene Therapy and Drug Targets. Web. 23 Aug. 2010. <http://www.discoverygenomics.net/sbts.html>.

Online webpage. Discovery Genomics, Inc., is a Minnesota-based company which works with the University of Minnesota in developing components for SB transposon systems. The SB transposon system was first developed by Professor Perry Hackett. A novel gene transfer system consists of the SB transposon, the SB transposase, and the therapeutic gene. The DGI therapeutic SB transposons are advantageous because they are non-viral, insulated to avoid activation of non-target genes, can be used in life-long therapy with reliable results, and they have the highest level of activity and application in a wide range of diseases. Validated technology consists of the ability to mediate therapeutic levels of gene expression for long durations of time in mammals. The SBIR grant was formed to test the application of the system in Hemophilia A and ex-vivo delivery in Fanconi Anemia. There is a patent regarding SB transposon procedure which was filed in December of 2002 that is awaiting confirmation by the federal government.

Elion, Elaine A. "Detection of Protein-Protein Interactions by Coprecipitation." PubMed Central - Genome Biology. U.S. National Library of Medicine, National Institutes of Health, May 2006. Web. 04 Mar. 2011. <http://www.ncbi.nlm.nih.gov/pubmed/18428641>.

Lab report. Researchers from the University of Rochester Medical School and Department of Molecular Genetics and Microbiology have discussed the advantages associated with the employment of co-immunoprecipitation techniques in protein-protein interaction studies. First, as a form of protein affinity chromatography Co-IP can detect weak associations which cannot withstand standard purification methods involving substantial dilution of initial cell extra. Second, Co-IP tests for associations between proteins within whole-cell extracts where proteins are present in native concentrations among a complex mixture of cellular components. However, verification of authenticity must be established for Co-IP. Researchers must ensure the co-precipitated protein is precipitated by the antibody itself and not a contaminated antibody in preparation. While monoclonal antibodies can generally solve this problem, researchers must ensure the antibody does not itself recognize the co-precipitated protein as well.

Fujita, Tomoyuki, Jun Igarashi, Erin R. Okawa, Takahiro Gotoh, Jayanthi Manne, Venkatadri Kolla, Jessica Kim, Huaqing Zhao, Bruce R. Pawel, Wendy B. London, John M. Maris, Peter S. White, and Garrett M. Brodeur. "CHD5, a Tumor Suppressor Gene Deleted From 1p36.31 in Neuroblastomas." JNCI Oxford Journals 100.13 (2008): 940-49.PubMed. U.S. National Library of Medicine, National Institutes of Health, 2 July 2008. Web. 10 Dec.

2010. <http://www.ncbi.nlm.nih.gov/pubmed/18577749>.

Lab report. Neuroblastomas, a common childhood cancer characterized by sympathetic nervous system tumor growth, have been used in genomic analyses. Since 1977, several studies have characterized neuroblastomas by a deletion of the short arm of chromosome 1 (1p). This study noted 1p deletions in approximately 35% of all neuroblastomas and in 70%-80% of all high-risk tumors. These numbers imply the loss of function of a tumor suppressor within the region. The region in question is a 2-Mb region of 1p36.31. 1p36.31 has been mapped and shown to encode 23 independent genes. To compress this list of gene candidates, researchers analyzed expression of the 23 genes in neuroblastoma cell lines. When screened for several characteristics (low expression, preferential expression in the nervous system, etc.), the only candidate remaining was chromodomain helicase DNA-binding protein 5 (CHD5).

"Gene Therapy." Oak Ridge National Laboratory. U.S. Department of Energy Office of Science, Office of Biological and Environmental Research, Human Genome Program, 11 June 2009. Web. 22 July 2010. <http://www.ornl.gov/sci/techresources/Human_Genome/medicine/genetherapy.shtml>.

Online webpage. A functioning gene can be placed in a nonspecific location in a genome to replace a nonfunctional gene by homologous recombination, selective reverse mutation, and corrective regulation mechanisms. A vector is used to deliver the therapeutic gene to a patient’s target cells. Viruses have evolved a mechanism of delivering genes. Retroviruses (double-stranded DNA copies of RNA genomes), Adenoviruses (double-stranded DNA genomes), Adeno-Associated Viruses (single-stranded DNA genomes in chromosomes), and Herpes Simplex (double-stranded DNA) are examples. Non-viral options include direct introduction of therapeutic DNA, creation of synthetic lipid sphere with an aqueous core to pass through cell membranes, and chemically linking to a molecule that binds to cell receptors. Researchers are working to introduce a 47th chromosome into target cells to prevent immune system backfires. Some factors which limit gene therapy include immune responses, multigene disorders, and expression longevity.

Gould, David, Nasim Yousaf, Rewas Fatah, and Yuri Chernajovsky. "Full Text | Gene Therapy With An Improved Doxycycline-regulated Plasmid Encoding a Tumour Necrosis Factor Alpha Inhibitor in Experimental Arthritis." Arthritis Research & Therapy. BioMed Central Ltd, 25 Jan. 2007. Web. 23 July 2010. <http://arthritisresearch.com/content/9/1/R7>.

Lab report. Researchers from the University of London tested TNF-A molecules in arthritis treatments. Tumor Necrosis Factor Alpha inhibitors used with methotrexate were found to be the best agents for rheumatoid arthritis. Plasmid DNA, unlike virus DNA, is devoid of protein components and is non-immunogenic. This makes plasmids safe delivery vectors, but plasmid lack mechanisms for entering cells. Plasmids can transfect skeletal muscle (Wolf). When combined with electroporation, plasmid efficiency is enhanced 100-fold. Biological molecules, such as TNF-A, can be delivered via plasmids as encoding DNA. The two synthetic vectors in the experiment were pGTMIK and pGTTMIK. Regulated promoters enable the expression of therapeutic molecules to be reduced during disease remission cycles. Pharmacologically regulated systems of gene expression have been developed. One example is the tetracycline system which successfully uses bacterial components of tetracycline resistance in synthetic systems.

Glick, B.R., and J.J. Pasternak. Molecular Biotechnology Principles and Applications of Recombinant DNA. 3rd ed. Washington, D.C.: ASM, 2005. Online. 22 July 2010 <http://www.webbooks.com/MoBio/Free/Ch9A4.htm>

Online book excerpt. Cloning vectors can be accomplished in several systems. Plasmid vectors have a polylinker for recognition of several restriction enzymes, an ampicilin-resistant gene which codes for beta-lactamase for selective amplification, and replication origin for proliferation in host cells. Lambda phages, bacterial viruses, have 1000 times more efficiency over naked plasmids. The ends of Lambda phage DNA contains COS sites with 12 complementary nucleotides which bind together to form circular DNA in the presence of DNA ligase. When Lambda phages are constructed, Nu1 and A proteins recognize and prepare recombinant DNA in vitro. Cosmid vectors mix naked plasmid vectors with COS sites, allowing DNA insertion in Lambda phage heads. YAC systems make use of telomeres, centromeres, and autonomous replicating sequences. First, target DNA is partially digested by EcoRI and then a YAC vector is cleaved by EcoRI and BamHI. The cleaved segments and partially digested DNA fragments form an artificial chromosome in yeast cells for plasmid cloning.

Gollins, H., J. McMahon, K. E. Wells, and D. J. Wells. "Gene Therapy - High-efficiency Plasmid Gene Transfer Into Dystrophic Muscle." Nature Publishing Group : Science Journals, Jobs, and Information. Macmillan Publishers Limited, 04 Oct. 2002. Web. 23 July 2010. <http://www.nature.com/gt/journal/v10/n6/full/3301927a.html>.

Lab report. Researchers from the Imperial College Faculty of Medicine experimented with various methods for increasing the efficiency of plasmid transfer in skeletal tissue cells. Direct injection of plasmids into skeletal muscle is an easy way to transfer genes. Plasmid gene transfer is hampered by poor transfection efficiency. The inception of electrotranfser-mediated gene delivery has resulted in the largest improvement in efficiency. Electrotransfer-mediated gene therapy is being researched for muscular dystrophies, yet the process induces muscular damage. Skeletal muscle has a number of “walls” comprised of extracellular matrix. Myofibres are organized into bundles surrounded by ECM. The pretreatment of skeletal muscle with bovine hyaluronidase can improve transfection efficiency. This is the result of improved accessibility for plasmid DNA into the muscle fiber surface. However, even with bovine hyaluronidase pretreatment, transfection efficiency does not improve with higher levels of connective tissue.

Hackett, Christopher S., Aron M. Geurts, and Perry B. Hackett. "Predicting Preferential DNA Vector Insertion Sites: Implications for Functional Genomics and Gene Therapy." PubMed Central – Genome Biology. National Center for Biotechnology Information, U.S. National Library of Medicine, 20 Dec. 2005. Web. 23 Aug. 2010.

<http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2106846/?tool=pubmed>.

Lab report. Scientists from the University of Minnesota discussed known factors which determine therapeutic gene vector insertion sites. There are four potential genetic consequences of integration of transgenic cassettes into chromatin. Integration into heterochromatin will result in no expression. Integration into intergenic euchromatin leads to GOF. Integration into transcriptional regulatory euchromatin can lead to GOF and activate neighboring genes. Integration into a transcriptional unity can lead to GOF and LOF, DGF, and DNF of the host neighboring genes. Transposons are preferred to viral vectors to avoid a preference for integration into genes, purification of toxins, a potential to elicit unwanted immune responses, expense associated with production, and constraints on therapeutic size. The only negative tradeoff is difficulty in delivery. Hydrodynamic delivery is working to solve this problem.

Hackett, Perry B., David A. Largaespda, and Laurence JN Cooper. "A Transposon and Transposase System for Human Application." PubMed Central - Genome Biology. U.S. National Library of Medicine, National Institutes of Health, 26 Jan. 2010. Web. 26 Aug. 2010. <http://www.ncbi.nlm.nih.gov/pubmed/20104209>.

Lab report. Researchers from the University of Minnesota investigated nonviral approaches to therapeutic genetic delivery. Viral approaches are limited by high cost of manufacture and low number of production facilities. Clinical-grade DNA plasmids can be prepared at lower costs. Researchers have been working to improve integration efficiency by transient coexpression of a hyperactive transposase in transposon systems. Local insertional preferences are dependent on DNA structure and sequence. Factors including DNA type (B-DNA and A-DNA), A-philicity, bendability, and protein-induced parameters (twist, tilt, slid, roll, rise) can determine the attractiveness of an insertion site for vectors. Sleeping Beauty transposons target random TA sites while piggyBac transposons target gene-encoding TTAA sites for example. The report examined safety implications of application of the SB system to genetically modify T cells.

Jordan, TS. "Cryptic Plasmid Cancer Gene Therapy." EHow | How To Do Just About Everything! | How To Videos & Articles. LIVESTRONG, 2010. Web. 23 July 2010. <http://www.ehow.com/about_5575961_cryptic-plasmid-cancer-gene-therapy.html>.

Online webpage. A new form gene therapy known as “cryptic plasmids” has been recently developed. Targeted re-engineering of the body’s genetic code for cancer proliferation inhibition is one of the innovative techniques being developed to combat cancer. Cancer occurs when the body fails to properly contain and destroy abnormal cell growth. Abnormal growth is believed to be caused by the activation of oncogenes and inactivation of TSGs. Gene therapy is used to modify the genetic coding of cells, rendering them immune to disease. For cancer, however, gene therapy seeks convince the body’s immune system to destroy cancerous cells. Cryptic plasmids have no known effect on cells. They have no additional genetic material beyond that needed for cell reproduction. Cryptic plasmids are useful as a transport mechanism for new genetic material during gene therapy. They are a “blank slate” and ideal for re-engineering. Cryptic plasmids allow for a safe delivery allowing scientists to worry only about fixing the dysfunctional gene.

Kirkland, James B. "Poly ADP-ribose Polymerase-1 and Health." Experimental Biology and Medicine 235 (2010): 561-68. PubMed. U.S. National Library of Medicine, National Institutes of Health, May 2010. Web. 10 Dec. 2010. <http://www.ncbi.nlm.nih.gov/pubmed/20463295>.

Lab report. PARP-1 is associated with the vitamin niacin. Niacin, known as vitamin B3, is an organic compound (C6H5NO2) which is an essential human nutrient. According to this study, research concerning niacin picked up in the mid-1900s when the disease pellagra was discovered to be caused by niacin deficiencies. Niacin is required to form nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) used in cellular respiration and photosynthesis respectively. As information about niacin was compiled, information about the interaction of these compounds in oxidation-reduction reactions was compiled. These oxidation-reduction reactions are regulated by various enzymes, the most active being poly (ADP-ribose) polymerase-1 (PARP-1). PARP-1 has been shown to control processes including DNA repair and chromatin development.

Kraus, W. Lee. "PARP-1 And Cancer." Active Motif: Tools To Analyze Nuclear Function. Cornell University, 2010. Web. 14 Jan. 2011. <http://www.activemotif.com/parp1.html>.

Lab report. PARP-1 inhibitors have become the focus of medical cancer therapies. This clinical study explained the modern thesis of PARP-1 inhibition in cancerous cells. By targeting cells making use of the BRCA HR pathway, PARP-1 inhibitors can selectively impair DNA damage repair in cancer cells inducing cell lysis. This study noted activity with BRCA1 and BRCA2 deficiencies, commonly found in breast and ovarian cancer patients. These deficiencies prevent double-strand break repair capacity while PARP-1 inhibitors impair single-strand break repair. These two functions in turn advance the number of double-strand breaks in cancer cells. However, animal models have shown that low niacin levels and inhibition of PARP-1 function can lead to the development of leukemias. New research is focused on searching for a solution which can restrict PARP-1 synthesis while preventing NAD+ depletion.

Largaespada, David A. "Transposon-mediated Mutagenesis of Somatic Cells in the Mouse for Cancer Gene Identification." PubMed Central - Genome Biology. U.S. National Library of Medicine, National Institutes of Health, 02 July 2009. Web. 26 Aug. 2010. <http://www.ncbi.nlm.nih.gov/pubmed/19607923>.

Lab report. Researchers at the University of Minnesota employed synthetic transposons for cancer gene identification. SB is a member of the Tc1/Mariner family of DNA transposable elements which integrate at “TA” dinucleotides. SB was originally identified as a long-dormant transposable element in salmon fish. The report hypothesized that the SB transposon could be used to “tag” cancer genes in mouse somatic cells near T2/Onc. The SB transposase enzyme recognizes binding sites within the IRs excises and then integrates elsewhere at a TA dinucleotide. The excision site is repaired by the host cell machinery leaving behind a 3 bp footprint, CAG or CTG. In the case of tumor suppressor genes, both alleles must be inactivated for cancer to occur generally/ T2/Onc mobilization could identify tumor suppressor genes when they showed haplo-insufficiency for tumor suppression. If the two alleles were inactivated by two independent insertions, or if the second was inactivated by other means after the first was inactivated.

Lew, Denise, John Norman, Terrie Latimer, Anna Abai, and Drake Laface. "Cancer Gene Therapy Using Plasmid DNA: Pharmacokinetic Study of DNA Following Injection in Mice : Deep Blue at the University of Michigan." Home : Deep Blue at the University of Michigan. University of Michigan Medical Center, 1995. Web. 23 July 2010. <http://deepblue.lib.umich.edu/handle/2027.42/63149>.

Lab report. Researchers at the University of Michigan in 1995 tested whether an injection of MHC class I molecules would activate cancer detection by human immune systems. Direct intratumoral of plasmid DNA encoding foreign antigens or cytokines was employed. A majority of malignancies come up in immunocompetent hosts. Tumors escape host defenses by avoiding expression of major histocompatibility complex class I molecules on cancerous cell surfaces. Isolated cells from natural tumors lack MHC class I antigens. Studies show that direct transfer of MHC class I molecules into tumors can facilitate recognition by the immune system. However, this process requires in vitro cell cultures from human patients to avoid tissue rejection. The intravenous route of plasmid DNA injection provides the greatest potential for detection of systemic expression of and manifestation of systemic toxicity. Studies in mice have shown the overall safety of IV DNA-cationic lipid injection as an in vivo gene transfer methodology.

McCooey, Beth, and Matthew Kuure-Kinsey. "An Introduction to Recombinant DNA." Rensselaer Polytechnic Institute (RPI) :: Architecture, Business, Engineering, IT, Humanities, Science. Rensselaer Polytechnic Institute, 2000. Web. 23 July 2010.

<http://rpi.edu/dept/chem-eng/BiotechEnviron/Projects00/rdna/rdna.html>.

Online webpage. Much research has been done concerning recombinant DNA, discovering what rDNA is, how rDNA is produced, and how rDNA functions. Recombinant DNA forms when one piece of DNA is matched with another. During microinjection, DNA is injected directly into the nucleus. During biolistics, host cells are bombarded with high velocity microporjectiles. Phage introduction makes use of viruses over bacteria. During in vitro packagings, a vector is used with a Lambda or MI3 phage to produce plaques with recombinants. rDNA is successful when a host cell produces one or more proteins coded by the rDNA. The protein is not produced unless expression factors are added with instructions for DNA transcription. When the gene has introns or bacterial terminator signals, problems can occur. Animal cells are hard to work with because they need a solid support surface and have complex growth needs.

Miller, J.F. "BACTERIAL PATHOGENESIS. PART A. IDENTIFICATION AND REGULATION OF VIRULENCE FACTORS." Bacterial Transformation by Electroporation. Vol. 235. 375-85. Methods in Enzymology. CSA Illumnia. ProQuest. Web. 30 June 2010. <http://md1.csa.com/partners/viewrecord.php?requester=gs&collection=ENV&recid=3973186&q=bacterial+transformation+techniques&uid=789602431&setcookie=yes>.

Online book excerpt. Electroporation is a fundamental method employed in genetic engineering. During electroporation, DNA is introduced into bacteria by the application of a high-voltage pulse to a sample of cells for induction of transient membrane permeability and DNA uptake. The mechanism by which electroporation induces reverse membrane permeability has not been wholly elucidated. Nevertheless, electroporation has been shown to be successful in 100 bacterial species, gram positive organisms, gram negative organisms, acid-fact organisms, and known human and animal pathogens. This process is commonly used in animal, plant, fungal, and protozoan cells. The efficiency of electroporation varies widely among organisms. An optimized technique produces 10 transformants/?g of plasmid DNA in E. Coli cells. Low efficiency among specific individuals can be improved by a systematic evaluation of several key variables, however, some organisms are completely intractable to DNA uptake.

Oesterreich, S., and S.A. W. Fuqu. "Tumor Suppressor Genes In Breast Cancer." Endocrinology Journals. Endocrine-Related Cancer, 1999. Web. 10 Jan. 2011. <http://erc.endocrinology-journals.org/cgi/reprint/6/3/405.pdf>.

Online webpage. Researchers have discovered more about cancer’s biochemical mechanisms. Cancer is caused by a collection of environmental, hereditary, and physical factors. Cancer is caused on a genetic level by the activation of mutated oncogenes which regulate normal cell growth or the inactivation of various tumor suppressor genes (TSGs) which act as “brakes” on cell growth in human cell systems. The inactivation of TSGs can be explained by “Knudson’s Hypothesis.” Mutation of an oncogenic allele will not necessarily lead to cancer because normally functioning TSGs will counterbalance this impetus. Only a growth impetus from an activated oncogene and a damaged TSG will lead to unchecked cancer proliferation. Cancer research has diverged, one group of scientists searching for cancer-causing genes and another side searching for tumor-suppressor genes.

O'Gara, Maureen. "Successful Synthetic Genome Introduces New Age of Biologic Tinkering: Vision.Org | SYS-CON MEDIA." Home | SYS-CON MEDIA. Marketwire, 22 June 2010. Web. 22 July 2010. <http://www.sys-con.com/node/1440338>.

Lab report. The J. Craig Venter Center recently manufactured a bacterium completely powered by a synthetic chromosome, characterized as the world’s first synthetic organism. Coupled with advances in the Human Genome project, synthetic biology gives scientists power to mold the processes of life. The chromosome of bacterial species Mycroplasm mycoides was sequenced in a digital file. The file was then edited by scientists. Several processes of gene discovery and recombinant DNA live can be discovered with the power to manipulate and invent genomes for use in synthetic bacterium. The J., Craig Venter Center readily expects synthetic bacteria to be helpful in pinpointing enzymatic pathways. Development of more complex processes by which synthetic organisms can be created will expand our biotechnical power. This new power comes doesn’t come with concerns however, and many ethical reservations will have to be addressed before new techniques are developed.

"Plasmids and Lambda Phage Are Choice Vectors for DNA Cloning in Bacteria." BIOWORLD. Biology Science of the World, 07 Mar. 2010. Web. 22 July 2010. <http://bioscienceofworld.blogspot.com/2010/03/plasmids-and-lambda-phage-are-choice.html>.

Online webpage. BIOWORLD, an American chemical company, has described several choice vectors for DNA cloning in bacteria. pBR322 codes for tetracycline and ampicillin resistance. Endonucleases cleave pBR322. Insertion of DNA at EcoRI doesn’t modify insert genes, but insertion at HindIII, SalI, or BamHI inactivates genes coding for tetracycline resistance. Lambda phages may be used. In a lytic pathway, viral DNA and proteins are inserted, produced, and packaged before cell lysis. In a lyso-genic pathway, viral DNA is inactively inserted into the host genome and replicated, activating with environmental signals. Mutant LGT-Lb only has two EcoRI cleavage sites instead of five. A DNA insert (10 kb) between the two ends of L DNA enables recombinant DNA to be packaged. M13 phages are useful for sequencing inserted DNA. A protein coat of 2710 subunits allows the virus to enter E. coli through a sex pilus. Only the + DNA strand is packaged into the new viruses. M13 is produced by cleaving the circular RF DNA at one restriction site.

Phizicky, E. M. "Co-Immunoprecipitation (Co-IP)." Protein Purification, Modification and Detection: Pierce Protein Research. Thermo Fisher Scientific, Inc., 2002. Web. 03 Mar. 2011. <http://www.piercenet.com/browse.cfm?fldID=9C471132-0F72-4F39-8DF0-455FB515718F>.

Online webpage. Co-immunoprecipitaiton is one of the most widely used methods for protein-protein interaction analyses. The technique is a more complex version of immunoprecipitation used for antigen detection and purification focused on identifying antigen interacting proteins. In immunoprecipitation, an antibody against a specific target protein will form an immune complex with the target. The immune complex is captured or precipitated on a beaded support to which an antibody-binding protein becomes immobilized with proteins not precipitated by the beads (such as protein A or G) washed away. Finally, the antigen is eluted from the support and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by Western blot detection for verifying antigen identity. For Co-IP, nonspecific binding to IP components and antibody contamination can make identifying physiological protein-protein interactions difficult.

Phizicky, Eric M., and Stanley Fields. "Protein-Protein Interactions: Methods for Detection and Analysis." PubMed Central - Genome Biology. U.S. National Library of Medicine, National Institutes of Health, Mar. 1995. Web. 04 Mar. 2011. <http://www.ncbi.nlm.nih.gov/pubmed/7708014>.

Lab Report. Researchers from John Wiley & Sons, Inc., have compiled a standardized co-precipitation procedure for protein-protein interaction analyses. For protein detection, the use of a short peptide or epitope recognized by commercial high-affinity monoclonal antibodies is advised. The epitope, usually influenza hemagglutinin protein (HA) and human c-Myc, is connected along the amino terminus of the polypeptide. Proteins can be fused to small proteins with high affinity to small molecules which can be connected to a solid support. This approach is valuable when a protein is to be precipitated with immunoglobin heavy or light chains in an SDS-polyacrlymide gel. Next, whole-cell extracts which optimize the yield and activity of proteins under analyses need to be developed. By collecting glutathione-agarose or glutathione-Sepharose beads, protein A/G slurries may be performed for protein identification.

"Recombinant DNA Technology Has Revolutionized All Aspects of Biology." BIOWORLD. Biology Science of the World, 07 Mar. 2010. Web. 22 July 2010. <http://bioscienceofworld.blogspot.com/2010/03/recombinant-dna-technology-has.html>.

Online webpage. BIOWORLD, an American chemical company, explains scientific applications of recombinant DNA technology. With recombinant DNA, DNA fragments are covalently joined to a DNA vector which can replicate autonomously in a host. The vector is prepared for accepting new DNA fragments by cleaving near specific sequences with restriction enzymes. The cuts produce complementary single-strand “sticky ends” in which the insert DNA can be placed. The single-stranded ends of the fragment are complementary to the plasmid and can be sealed by DNA ligase. DNA ligase catalyzes the formation of a phosphodiester bond at a break in the DNA chain. ATP or NAD+ is needed for the reaction as well as a free 3-hydroxyl and 5-phosphoryl group. This method can be shortened using a chemically synthesized DNA linker which can be cleaved by restriction enzymes. “Sticky ends” are formed for the DNA fragment, not the other way around.

Somia, Nikunj V., Hiroyuki Miyoshi, Mark J. Schmitt, and Inder M. Verma. "Retroviral Vector Targeting to Human Immunodeficiency Virus Type 1-Infected Cells by Receptor Pseudotyping." The Journal of Virology. American Society for Microbiology, 21 Jan. 2000. Web. 24 Aug. 2010. <http://jvi.asm.org/cgi/content/full/74/9/4420>.

Lab report. Researchers from the University of Minnesota designed retroviral vectors for in vivo delivery applications. Competent retroviruses are constructed using a packaging cell line and vector. The former provides proteins needed to assemble retroviruses (products of the GAG, POL, and ENY genes). The vector consists of a therapeutic gene and retroviral packaging signal bound by terminal repeats. The tropism of the retrovirus is dictated by the product of the ENY gene. Commonly used packaging lines develop an ecotropic envelope restricting infection to rodent cells or an amphotropic envelope allowing infection of most mammalian cells. For in vivo gene delivery scientists must incorporate targeting molecules into the virion membranes or change envelope proteins. Scientists can engineer a ligand or an antibody binding site for a cell antigen with a cognate receptor onto the envelope or exploit a recognition protein from a virus with evolved tropism.

Somia, Nikunj V., Monica Zoppe, and Inder M. Verma. "Generation of Targeted Retroviral Vectors by Using Single-Chain Variable Fragment: an Approach to in Vivo Gene Delivery."Proceedings of the National Academy of Sciences. The National Academy of Sciences of the United States of America, 11 May 1995. Web. 24 Aug. 2010.

<http://www.pnas.org/content/92/16/7570.full.pdf html>.

Lab report. Researchers under Dr. Nikunj Somia from the University of Minnesota experimented with a new method of receptor pseudotyping in viral vectors. Psuedotyping with viral molecules has been the most successful approach in generating high-titer vectors, but a “redesign” approach has only generated low-titer vectors. This is due to the uncoupling of binding proteins and membrane fusion in the designed envelopes. This is naturally coupled in evolved envelopes. A study found that the cell surface glycoprotein CD4 could be incorporated into avian leukosis virus, the primary receptor for human immunodeficiency type 1. However this pseudotyped virus could not infect cells expressing HIV-1 proteins gp120 and gp41 because a co-receptor protein was needed. Murine leukemia viruses were tested using a cell line which only expresses the gag and pol genes of MLV and transduces the gene for green fluorescent protein. Pseudotyping of virus envelopes needs to be tested as membrane envelopes are not necessarily interchangeable.

Summers, D.K. Introduction. The Biology of Plasmids. Oxford UP, 1996. Online. 22 July 2010 <http://science.jrank.org/pages/48703/Plasmids.html>.

Online book excerpt. Plasmids are extrachromosomal DNA found mainly in bacterial species. During the 1950s, plasmids were discovered. Scientists believed plasmids were responsible for antibiotic resistance in bacteria. The origin of plasmids is unknown, but they are thought to have evolved from bacterial phages. Plasmids naturally propagate by infections transfer among bacteria, some only being able to spread among one species while others being able to spread throughout a “horizontal” gene pool of unrelated species. While small plasmids have a few hundred base pairs and a replicon gene cluster for replication, large plasmids have several hundred kilobases and genes which regulate plasmid maintenance or code for host functions. Plasmids do not however have gene sequences which are categorizable with other phylogenetic organisms. They are viewed as cooperators because they provide helpful functions to a host to resist environmental pressures.

Thisted, T., N.S. Sorensen, and K. Gerdes. Mechanism of Post-Segregational Killing I. Vol. 13. 1994. Print. EMBO Journal (1960-1968). 22 July 2010. <http://science.jrank.org/pages/48703/Plasmids.html>.

Online book excerpt. In 1995, an experiment was conducted to investigate the impact of hok and sok genes within plasmids to conduct post-segregational killing (PSK), shaping our modern understanding of the process. During PSK, daughter cells without specific plasmids commit apoptosis following round one of division. The system is prevalent among R1 enterococcal plasmids with characteristic low copy and high plasmid loss numbers. Hok genes code for lethal toxins while sok genes code for an “antidote,” antisense RNA which binds and disables hok DNA. Because unstable sok gene RNA is quickly dispensed from plasmid-free bacteria, many scientists believe PSK has evolved among bacteria to reduce local competition and enhance the fitness of relatives with R1 plasmids. RM systems function in much of the same way, making use of two component gens and a restriction enzyme R to cut individual DNA sequences for bacterial methylation.

Thomas, C.M., ed. The Horizontal Gene Pool. Amsterdam, 2000. Science Encyclopedia. Net Industries. Web. 30 June 2010. <http://science.jrank.org/pages/48703/Plasmids.html>.

Online book excerpt. Because plasmids exact energy from bacterial hosts, natural selection favors bacteria with low copy and plasmid numbers. This helps explain the evolution of several bacterial processes, including PSK systems and conjugation. Conjugation is a form of sexual reproduction for bacteria. During conjugation, plasmids are transferred from one bacterial cell to another with the help of one of 30 known plasmid genes. This undirected process begins with the development of the sex pilus, a 10 nm diameter hollow tube which contacts potential recipients. The sex pilus protects plasmid DNA from harmful environmental conditions outside of bacterial cells. Bacterial cells will reject contact unless they have plasmids from different compatibility groups in order to foster genetic diversity. Some bacteria actively search for partners who are marked “plasmid-free” by secreted pheromones which foster the production of a sex pilus. Plasmids will suppress pheromone production to prevent conjugation. Conjugation allows for vast genetic diversity among bacteria.

Tu, Anh-Hue T. "Transformation of Escherichia Coli Made Competent by Calcium Chloride Protocol."Microbe Library. Microbe Library. American Society for Microbiology, 28 Sept. 2008. Web. 30 June 2010. <http://www.microbelibrary.org/edzine/details.asp?id=2794&Lang=>.

Lab report. A “calcium chloride” technique can be used for DNA uptake in E. coli cells. This chemical method uses divalent and multivalent cations (calcium, magnesium, manganese, rubidium, and hexamine cobalt) in the process. E. coli has one long O-linked polysaccharide which blocks DNA from entering the cell. The cell envelope in E. coli is made up of an outer membrane, inner membrane, and cell wall. The outer cell membrane is made up of phospholipids, proteins, and lipopolysaccharides. Channels between the outer and inner cell membrane allow for the transport of DNA, but negatively charged DNA fragments are repelled by negatively charged macromolecules on the outer surface of the cell membrane. Adding CaCl2 neutralizes outer membrane layer polyanions. Competent cells are placed on ice for stabilization of lipid membrane. By heat shock, the fluidity of a semi-crystalline membrane state is modified. Magnesium is supplemented to enhance membrane ionic interactions.

“UCR Entomologists To Develop Special Bacteria To Combat Spread Of Mosquito-Borne Diseases." Medical News Today: Health News. University of California - Riverside, 16 June 2010. Web. 21 July 2010. <http://www.medicalnewstoday.com/articles/191865.php>.

Online webpage. Researchers from the University of California have undertaken to combat mosquito-borne diseases. Brian Fedreici will use a five-year, $1.86 million grant to develop genetically engineered bacteria to kill mosquito larvae. This engineered bacteria will be designed to produce proteins which are highly toxic to only mosquitoes, a hybrid of B. sphaericus and B. thuringiensis bacterial strains. The recombinant is available in dried powder or in suspended liquid. The researchers cloned the genes responsible for killing mosquitoes and used DNA plasmids for insertion by bacterial transformation. Mosquitoes can become resistant to normal control agents, but with evolutionary recombinant plasmids resistant takes longer to develop. The University of California science department will focus on the evolution of insecticide resistance to microbial toxins, the genetic basis of this evolutionary resistance, and conduct an evaluation of the bacterial insecticides. By reducing adult female longevity, scientists can reduce overall mosquito numbers.

Watson, James D., and Andrew Berry. "DNA, Dollars, Drugs." DNA: the Secret of Life. New York: Alfred A. Knopf, 2003. 115-16. Print.

Book excerpt. Bacteria have no mechanism to deal with introns, non-coding segments of DNA, because they lack the capacity to edit mRNA, lacking the enzymes which do so. This problem is what made inducing a bacterium to produce insulin for scientists very challenging. Working for decades, scientists finally made the process cheap and fast. The European company Biogen used DNA from human cells to employ a new discovery. The central dogma which states that DNA needs to make RNA for use in cells was broken with the discovery of a species of viruses in the 1950s. These viruses could convert RNA into DNA when inserted into host cells with the enzyme reverse transcriptase. Biogen needed to isolate mRNA which coded for the human insulin gene without an intron and then convert this strand into DNA during the process. DNA is more workable than RNA because of a slower rate of decay.

Watson, James D., and Andrew Berry. "Playing God." DNA: the Secret of Life. New York: Alfred A. Knopf, 2003. 115-16. Print.

Book excerpt. By the 1970s, the components for a recombinant DNA procedure were in place. First, DNA molecules could be cut with restriction enzymes allowing scientists to isolate gene sequences. These sequences could be “glued” into plasmids by the enzyme DNA ligase. These “plasmid floppies” could finally be transplanted into bacterial cells by bacterial transformation. Bacterial cell division would then replicate the plasmid along with the cell’s own bacterial DNA allowing for the production of thousands of copies of DNA sequences. Herb Boyar’s labs in San Francisco were the first to produce a hybrid “recombinant” from two plasmids in E. coli cells. Unaltered plasmids can be cut with restriction enzymes and mixed in test tubes with the enzyme DNA ligase. Some plasmids would revert back into original form while other plasmids would take up individual DNA sequences with base pair homology with the split ends of the linearized plasmids. Boyar’s labs produced bacteria with tetracycline and kanamycin resistance.

Webster, Robert G., George Kemble, and Harry B. Greenberg. "Rescue of Influenza B Virus from Eight Plasmids." Proceedings of the National Academy of Sciences. PNAS, 2002. Web. 23 July 2010. <http://www.pnas.org/content/99/17/11411.full?sid=7daf8688-0cea-4725-9b606713c9bcb2d2>.

Lab Report. Researchers from the University of Tennessee developed a new method to produce vaccines for influenza B virus by using a plasmid-only reverse genetic system. This plasmid-only reverse genetic system facilitates production of vaccine viruses without the time needed for co-infection and selection procedures. 8 viral cDNAs of influenza B/Yamanashi/166/98 were cloned. Hemaglutinin (HA) and neuraminidase (NA) segments can be found in modern vaccines New antigenic variants have changed glycoprotein levels by nucleotide misincorporation. B/Yamagata/16/88 and B/Victoria/2/87 are lineages circulating in humans. Instead of finding a wild-type strain or using classical reassortment (coinfection with HA and NA level viruses and a biologically compatible virus) plasmids were used. There are several problems however. 14642 nucleotides need to be cloned. Influenza A and B are distinct, and the GenBank for influenza B is incomplete as well.

Wiesner, Stephen M., Stacy A. Decker, Jon D. Larson, Katya Ericson, Colleen Forster, Jose L. Gllardo, Chunmei Long, Zachary L. Demorest, Edward A. Zamora, Walter C. Low, Karen SantaCruz, David A. Largaespada, and John R. Ohlfest. "De Novo Induction of Genetically Engineered Brain Tumors in Mice Using Plasmid DNA." Cancer Research. American Association for Cancer Research, 15 Jan. 2009. Web. 26 Aug. 2010. <http://cancerres.aacrjournals.org/content/69/2/431.full>.

Lab report. Researchers from the University of Minnesota observed how well the SB transposon system fostered giloma growth. The most widely used model of malignant giloma involves intracerebral transplantation of cultures giloma cells. Spontaneous models of MG in which the tumor evolves with the host immune system are sought with the expectation that they will more accurately emulate human disease progression and predict clinical effectiveness. Hybrid spontaneous models were created using GEM that express a receptor for a replication-competent ALV splice acceptor avian retrovirus. This GEM has been bred to a second GEM expressing firefly luciferase in mitotic cells to allow cell division to be noninvasively monitored with bioluminescent imaging. Several investigators have achieved nonviral transfection of the murine brain with polyethylenimine/plasmid DNA complexes. Unfortunately, gene expression after plasmid DNA transfection is often transient. SB transposons can achieve chromosomal integration and long-term integration.

Wilber, Andrew, Joel L. Frandsen, Jennifer L. Geurts, David A. Largaespada, Perry B. Hackett, and R. Scott McIvor. "RNA as a Source of Transposase for Sleeping Beauty-Mediated Gene Insertion and Expression in Somatic Cells and Tissues." Nature Publishing Group. AGORA, HINARI, OARE, INASP, CrossRef and COUNTER, 20 Dec. 2005. Web. 23 Aug. 2010. <http://www.nature.com/mt/journal/v13/n3/abs/mt200675a.html>.

Lab report. Researchers from the University of Minnesota experimented with RNA as a source of transposase for the SB transposon system. Using DNA as a deliverable molecule is problematic because or re-integration and genotoxicity. The chance that RNA undergoes reverse transcription followed by cDNA integration is very unlikely. mRNA is advantageous for the following reasons. There is improved control with respect to the duration of transposase expression, minimal persistence in tissue, and a lowered chance of transgenic re-mobilization. A RNA sequence is incapable of integrating into the host genome as well. Scientists used a genomic recovery procedure to check experimental chromosomes and confirm that the new frequency of stable gene insertion was a result of transposition instead of random recombination. After sequencing the recovered plasmids, they revealed integration and confirmed use of mRNA in SB-mediated transposition.

Wilson, Matthew H., Craig J. Coates, and Alfred L. George. "Molecular Therapy – PiggyBac Transposon-Mediated Gene Transfer in Human Cells." Nature Publishing Group.Vanderbilt University, 29 Aug. 2006. Web. 22 July 2010. <http://www.nature.com/mt/journal/v15/n1/full/6300028a.html>.

Lab Report. Researchers from Vanderbilt University observed transposition rates of piggyBac and Sleeping Beauty transposon systems. Transposons are mobile genetic elements used to integrate transgenes into host genomes. PiggyBac systems has been used in insects and mice, however, researchers were testing the system against Sleeping Beauty, another system used in preclinical studies in humans. PiggyBac showed twice the efficiency with no overproduction inhibition. The piggyBac element is divided to insert a transgene between inverted repeats and asymmetric internal repeats to allow for “copy and paste” methods of transport. PiggyBac was shown to deliver large transposable elements without reduction in efficiency in mouse germlines. Sleeping Beauty is the widely used transposon system for preclinical gene therapy studies, a member of the Tc1/mariner family from the fish genome. SB is limited by overproduction inhibition, which occurs with higher transposase expression.

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