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A Novel Mechanism for Site-Directed Mutagenesis of Large Catabolic Plasmids Using Natural Transformation

Description: Natural transformation is the process by which cells take up DNA from the surrounding medium under physiological conditions, altering the genotype in a heritable fashion. This occurs without chemical or physical treatment of the cells. Certain Acinetobacter strains exhibit a strong tendency to incorporate homologous DNA into their chromosomes by natural transformation. Transformation in Acinetobacter exhibits several unique properties that indicate this system's superiority as a model for transformation studies or studies which benefit from the use of transformation as an experimental method of gene manipulation. Pseudomonas putida is the natural host of TOL plasmids, ranging between 50 kbp and 300 kbp in size and encoding genes for the catabolism of toluene, meta-toluate, and xylene. These very large, single-copy plasmids are difficult to isolate, manipulate, or modify in vitro. In this study, the TOL plasmid pDKR1 was introduced into Acinetobacter calcoaceticus strains and genetically engineered utilizing natural transformation as part of the process. Following engineering by transformation, the recombinant DNA molecule was returned to the native genetic background of the original host P. putida strain. Specific parameters for the successful manipulation of large plasmids by natural transformation in Acinetobacter were identified and are outlined. The effects of growth phase, total transforming DNA concentration, transforming DNA conformation, and gene dosage on transformation efficiency are presented. Addition of Acinetobacter plasmid DNA sequences to the manipulated constructs did not have an effect on transformation rates. Results suggest that a broadly applicable and efficient method to carry out site-directed genetic manipulations of large plasmids has been identified. The ability to easily reintroduce the recombinant DNA molecules back into the original host organism was maintained.
Date: August 2001
Creator: Williamson, Phillip C.
Partner: UNT Libraries

Construction of a Cloning Vector Based upon a Rhizobium Plasmid Origin of Replication and its Application to Genetic Engineering of Rhizobium Strains

Description: Rhizobia are Gram-negative, rod-shaped, soil bacteria with the ability to fix atmospheric nitrogen into ammonia as symbiont bacteroids within nodules of leguminous plant roots. Here, resident Rhizobium plasmids were studied as possible sources of components for the construction of a cloning vector for Rhizobium species.
Date: May 1992
Creator: Jeong, Pyengsoo
Partner: UNT Libraries

Gene Patents: A Brief Overview of Intellectual Property Issues

Description: This report is a brief discussion of the ethical, legal, and economic issues of gene patenting. The courts have upheld gene patents that meet the criteria of patentability defined by the Patent Act. However, the practice of awarding patents on genes has come under intense scrutiny by some scientists, legal scholars, politicians, and other experts.
Date: October 3, 2006
Creator: Schacht, Wendy H.
Partner: UNT Libraries Government Documents Department

Genetically Engineered Salmon

Description: This report discusses the genetically modified salmon. The term “genetic modification” refers to changes in an organism’s genetic makeup that do not occur in nature. Also, if approved by the Food and Drug Administration (FDA), Atlantic salmon would be the first genetically engineered (GE) animal to be marketed in the United States for human consumption.
Date: April 30, 2014
Creator: Upton, Harold F. & Cowan, Tadlock
Partner: UNT Libraries Government Documents Department

Agricultural Biotechnology: Background, Regulation, and Policy Issues

Description: This report discusses on going issues regarding biotechnology, which refers primarily to the use of recombinant DNA techniques to genetically modify or bioengineer plants and animals. Ongoing policy issues include the impacts of genetially engineered (GE) crops on the environment (e.g., pest and weed resistance), whether GE foods should be labeled, their potential contamination of conventionally raised and organic plants, and issues of liability.
Date: July 20, 2015
Creator: Cowan, Tadlock
Partner: UNT Libraries Government Documents Department

Agricultural Biotechnology: Background, Regulation, and Policy Issues

Description: This report discusses on going issues regarding biotechnology, which refers primarily to the use of recombinant DNA techniques to genetically modify or bioengineer plants and animals. Ongoing policy issues include the impacts of genetially engineered (GE) crops on the environment (e.g., pest and weed resistance), whether GE foods should be labeled, their potential contamination of conventionally raised and organic plants, and issues of liability.
Date: April 3, 2013
Creator: Cowan, Tadlock
Partner: UNT Libraries Government Documents Department

Genetic Exceptionalism: Genetic Information and Public Policy

Description: This report provides an overview of the nature of genetic information and its implications for individuals, family, and society. Individuals utilize genetic information to guide health care and other decisions, when possible, and may experience anxiety as a result of genetic test results. Genetic test results for an individual may often be informative for other close family members and thus influence their care decisions. Society must grapple with the effect genetic information may have on our conception of disease, as well as its impact on issues like privacy and equity. The report ends by summarizing the main policy issues involved with a genetic exceptionalist approach to public policy, including defining genetic information; physically separating genetic information from other medical information; unintended disparities between “genetic” and “nongenetic” disease; and the effect of legislation on participation in genetic research, on uptake of genetic technology and on the delivery of high quality health care.
Date: February 14, 2008
Creator: Sarata, Amanda K.
Partner: UNT Libraries Government Documents Department

New developments in biotechnology: field-testing engineered organisms: genetic and ecological issues: contractor documents, volume 2

Description: This report includes these topics: Ecological issues relevant to environmental applications of genetically altered organisms / Elliott A. Norse -- An ecosystems approach to potential perturbations of energy flow and nutrient cycles associated with environmental applications of genetically altered organisms / David C. Coleman and Robert E. Hodson -- Ecological impact of genetically engineered organisms on ecosystems / James R. Gosz, C.N. Dahm, and Patrick W. Flanagan -- The genetic basis of changes in host range or habitat / Adrianne Massey and Fred Gould.
Date: December 2, 1986
Creator: United States. Congress. Office of Technology Assessment
Partner: UNT Libraries Government Documents Department

Final Report: The DNA Files: Unraveling the mysteries of genetics, January 1, 1998-March 31, 1999

Description: The DNA Files is an award-winning radio documentary series on genetics created by SoundVision Productions. The DNA Files was hosted by John Hockenberry and was presented in documentary and discussion format. The programs covered a range of topics from prenatal and predictive gene testing, gene therapy, and commercialization of genetic information to new evolutionary genetic evidence, transgenic vegetables and use of DNA in forensics.
Date: May 1, 1999
Creator: Scott, Bari
Partner: UNT Libraries Government Documents Department

Genetic Modification of Fatty Acid Profiles in Cotton

Description: The industrial uses of cottonseed oil are limited by its fatty acid composition. Genetic modification of cotton lipid profiles using seed-specific promoters could allow cotton growers to produce valuable new oils in the seed without adverse effects on fiber quality and yield, therefore making this crop more commercially profitable. Transgenic cotton callus harboring a diverged fatty acid desaturase gene (FADX) from Momordica charantia was characterized for production of alpha-eleostearic acid (conjugated double bonds: 18:3 D9 cis, 11 trans, 13 trans), not normally found in cotton. Gas chromatography (GC) in conjunction with mass spectrometry (MS) confirmed production of alpha-eleostearic acid in the transgenic cotton tissues. A second series of transformation experiments introduced the cotton fatty acid thioesterase B (FATB) cDNA, fused to the seed-specific oleosin promoter into cotton to promote the over-expression of FATB, to generate cotton with increased palmitate in the cottonseed. PCR amplification, as well as fatty acid analysis by gas chromatography, confirmed introduction of the FATB cDNA in transgenic tissues. Collectively, these results demonstrate the feasibility of manipulating the fatty acid composition in cotton via transgenic approaches and form the basis for continued efforts to create novel oils in cottonseed.
Access: This item is restricted to the UNT Community Members at a UNT Libraries Location.
Date: August 2005
Creator: Rommel, Amy A.
Partner: UNT Libraries

Advanced Gene Editing: CRISPR-Cas9

Description: This report describes a new gene editing technology, known as CRISPR-Cas9, with the potential to revolutionize genetic engineering and the biotechnology industry. The report then provides information on the potential economic benefits of the technology and identifies some issues for congressional consideration, including the regulation of current and future products, national security concerns, and ethical and societal issues surrounding the use of the technology.
Date: April 28, 2017
Creator: Gallo, Marcy E.; Sargent, John F., Jr.; Sarata, Amanda K. & Cowan, Tadlock
Partner: UNT Libraries Government Documents Department

DNA Sliding Clamps: Just the Right Twist to Load onto DNA

Description: Two recent papers illuminate a long sought step in DNA sliding clamp loading. One paper reveals the structure of the PCNA clamp wrapped around DNA--still open from being loaded--while a second paper discovers that the clamp may assist this process by forming a right-handed helix upon opening.
Date: October 24, 2005
Creator: Barsky, D & Venclovas, C
Partner: UNT Libraries Government Documents Department

CBM.DIAGB.03.10.LLNL.007 Final Report

Description: The purpose of this project was to construct a system for characterizing the threat potential of genomic sequences, specifically assembled draft genomes. New genomes are characterized by initially comparing them against already-sequenced genomes. If the new genome is determined to be from a high-threat species, detailed (forensic-level) characterization is done based on gene and SNP (Single Nucleotide Polymorphism) data comparisons with all other previously sequenced members of that high-threat species. New genomes are compared against a large set of known virulence and antibiotic-resistance genes and also compared against a large set of vectors that could be used for bacterial genetic engineering. Together, these analyses provide a comprehensive initial assessment of the most likely phylogenetic placement of a new genome, plus an assessment of the known-gene content and an indication of any possible bacterial genetic engineering utilizing vector-mediated techniques. This provides an initial threat potential summary based on high information content comparisons (e.g., thousands of genes, SNPs, and potential genetic engineering vectors) that can be used to guide subsequent operational response or more detailed laboratory characterization.
Date: March 30, 2011
Creator: Slezak, T & Torres, M
Partner: UNT Libraries Government Documents Department

FLP-mediated conditional loss of an essential gene to facilitate complementation assays

Description: Commonly, when it is desirable to replace an essential gene with an allelic series of mutated genes, or genes with altered expression patterns, the complementing constructs are introduced into heterozygous plants, followed by the selection of homozygous null segregants. To overcome this laborious and time-consuming step, the newly developed two-component system utilizes a site-specific recombinase to excise a wild-type copy of the gene of interest from transformed tissues. In the first component (the first vector), a wild-type version of the gene is placed between target sequences recognized by FLP recombinase from the yeast 2 μm plasmid. This construct is transformed into a plant heterozygous for a null mutation at the endogenous locus, and progeny plants carrying the excisable complementing gene and segregating homozygous knockout at the endogenous locus are selected. The second component (the second vector) carries the experimental gene along with the FLP gene. When this construct is introduced, FLP recombinase excises the complementing gene, leaving the experimental gene as the only functional copy. The FLP gene is driven by an egg apparatus specific enhancer (EASE) to ensure excision of the complementing cDNA in the egg cell and zygote following floral-dip transformation. The utility of this system is being tested using various experimental derivatives of the essential sucrose-proton symporter, AtSUC2, which is required for photoassimilate transport.
Date: December 2007
Creator: Ganesan, Savita
Partner: UNT Libraries

Development and testing of biosensors that quantitatively and specifically detect organic contaminants

Description: This is the final report of a two-year Laboratory-Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). The project sought to develop a more sensitive and less expensive method of detecting organic contaminants. Assaying complex environmental samples for organic contaminant content is costly and labor intensive. This often limits extensive testing. Sensitive microbial biosensors that detect specific organic contaminants in complex waste mixtures without prior separation from other waste components have been developed. Some soil microbes degrade organic compounds that contaminate the environment. These bacteria sense minute quantities of particular organic compounds then respond by activating genes encoding enzymes that degrade these molecules. Genetic manipulation of these gene regulatory processes has been employed to develop unique biosensors that detect specific organic compounds using standard biochemical assays. Such biosensors allow rapid, sensitive testing of environmental samples for selected organic contaminants. The cost of biosensor assays is at least 100-fold less than present methods, allowing more rapid and extensive testing and site characterization.
Date: July 1, 1996
Creator: Jackson, P.; Keim, P.; Kuske, C. & Willardson, B.
Partner: UNT Libraries Government Documents Department

Controlled production of cellulases in plants for biomass conversion. Annual report, March 11, 1997--March 14, 1998

Description: The goal of this project is to facilitate conversion of plant biomass to usable energy by developing transgenic plants that express genes for microbial cellulases, which can be activated after harvest of the plants. In particular, the feasibility of targeting an endoglucanase and a cellobiohydrolase to the plant apoplast (cell wall milieu) is to be determined. To avoid detrimental effects of cellulose expression in plants, enzymes with high temperature optima were chosen; the genes for these enzymes are from thermophilic organisms that can use cellulose as a sole energy source. During the past year (year 2 of the grant), efforts have been focused on testing expression of endoglucanase E{sub 1}, from Acidothermus cellulolyticus, in the apoplast of both tobacco suspension cells and Arabidopsis thaliana plants. Using the plasmids constructed during the first year, transgenic cells and plants that contain the gene for the E{sub 1} catalytic domain fused to a signal peptide sequence were obtained. This gene was constructed so that the fusion protein will be secreted into the apoplast. The enzyme is made in large quantities and is secreted into the apoplast. More importantly, it is enzymatically active when placed under optimal reaction conditions (high temperature). Moreover, the plant cells and intact plants exhibit no obvious problems with growth and development under laboratory conditions. Work has also continued to improve binary vectors for Agrobacterium-mediated transformation, to determine activity of E{sub 1} at various temperatures, and to investigate the activity of the 35S Cauliflower Mosaic Virus promoter in E. coli. 9 figs.
Date: June 1, 1998
Creator: Danna, K.J.
Partner: UNT Libraries Government Documents Department