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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: 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

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

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

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

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

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

A New Selectable Marker System for Genetic Studies of Bacteria: Final Report

Description: Genetic manipulations in bacteria currently rely on the introduction of antibiotic resistance genes into a bacterial strain; for those organisms that will be used for commercial or industrial applications, the genetic cassette encoding the antibiotic resistance is sometimes removed after selection. it is clear that if alternative technologies could obviate the need to introduce antibiotic resistance into bacteria, they would most certainly become a standard tool in molecular micriobiology for commercial, industrial as well as research applications. Here, they present the development of a novel genetic engineering technology based on toxin-antitoxin systems to modify bacterial genomes without the use of antibiotic resistance in the mutagenesis process. The primary goal is to develop antibiotic-free selection for genetically altered select agent pathogens. They are adapting the toxinc-antitoxin system to enable gene replacement in select agent pathogens since the NIH restrictions introducing antibiotic resistance into select agent pathogens have hindered research with select agent pathogens.
Date: March 18, 2011
Creator: Parsons, D; Tolmasky, M; Chain, P & Segelke, B W
Partner: UNT Libraries Government Documents Department

Impact of a Genetically Engineered Probiotic Therapy and IGF-1 Genomics in the PAHenu2 Mouse Model of PKU

Description: Absence of functional phenylalanine hydroxylase results in phenylketonuria (PKU). Viable treatments remain few, expensive and secondary conditions such as osteopenia occur in most PKU patients. Objective 1: Given the recently described roles of gut microbes to aid host digestion, an orally administered genetically engineered probiotic as the delivery vehicle for enzyme replacement therapy was created. The engineered probiotic, pHENOMMenal, produced phenylalanine ammonia lyase with significant production of trans-cinnamate (phenylalanine cleavage product) in vitro and resulted in a reduction of 515 μM in blood phenylalanine when fed to PKU animals for 14 days (from 2307µM ± 264µM to 1792µM ± 261µM, n = 6, P < 0.05). The control probiotic produced no change in blood phenylalanine. Thus, pHENOMMenal treatment in PKU mice demonstrated engineered microbes could compensate for a metabolic deficiency of the host. Objective 2: Evaluate the PAHenu2 mouse model of PKU for a genetic discrepancy causing ocular enlargement and delayed development observed only after the PAHenu2 mutation was crossed to the C57BL/6J mouse. When compared to healthy littermates, ELISA indicated a consistent but insignificant decrease in plasma IGF-1 and an increase in ocular IGF-1 in PKU animals. SNP screening demonstrated a differential inheritance of IGF-1 alleles in healthy and PKU animals based on PAH allele inheritance. Ocular and developmental phenotypes in the PAHenu2 colony match those described in previous IGF-1 studies. Understanding the IGF-1 inheritance discrepancy will enable better osteopenia research using PAHenu2 mice and allow breeding of a healthier mouse colony for continued research. Collectively the results from this work describe a new therapeutic approach for treatment of PKU as well as a better understanding of the PAHenu2 mouse model to study this disease.
Date: December 2015
Creator: Durrer, Katherine Elaine
Partner: UNT Libraries

A Comparison of the First Two Sequenced Chloroplast Genomes in Asteraceae: Lettuce and Sunflower

Description: Asteraceae is the second largest family of plants, with over 20,000 species. For the past few decades, numerous phylogenetic studies have contributed to our understanding of the evolutionary relationships within this family, including comparisons of the fast evolving chloroplast gene, ndhF, rbcL, as well as non-coding DNA from the trnL intron plus the trnLtrnF intergenic spacer, matK, and, with lesser resolution, psbA-trnH. This culminated in a study by Panero and Funk in 2002 that used over 13,000 bp per taxon for the largest taxonomic revision of Asteraceae in over a hundred years. Still, some uncertainties remain, and it would be very useful to have more information on the relative rates of sequence evolution among various genes and on genome structure as a potential set of phylogenetic characters to help guide future phylogenetic structures. By way of contributing to this, we report the first two complete chloroplast genome sequences from members of the Asteraceae, those of Helianthus annuus and Lactuca sativa. These plants belong to two distantly related subfamilies, Asteroideae and Cichorioideae, respectively. In addition to these, there is only one other published chloroplast genome sequence for any plant within the larger group called Eusterids II, that of Panax ginseng (Araliaceae, 156,318 bps, AY582139). Early chloroplast genome mapping studies demonstrated that H. annuus and L. sativa share a 22 kb inversion relative to members of the subfamily Barnadesioideae. By comparison to outgroups, this inversion was shown to be derived, indicating that the Asteroideae and Cichorioideae are more closely related than either is to the Barnadesioideae. Later sequencing study found that taxa that share this 22 kb inversion also contain within this region a second, smaller, 3.3 kb inversion. These sequences also enable an analysis of patterns of shared repeats in the genomes at fine level and of RNA editing by comparison ...
Date: January 20, 2006
Creator: Timme, Ruth E.; Kuehl, Jennifer V.; Boore, Jeffrey L. & Jansen, Robert K.
Partner: UNT Libraries Government Documents Department

TMTI Task 1.6 Genetic Engineering Methods and Detection

Description: A large number of GE techniques can be adapted from other microorganisms to biothreat bacteria and viruses. Detection of GE in a microorganism increases in difficulty as the size of the genetic change decreases. In addition to the size of the engineered change, the consensus genomic sequence of the microorganism can impact the difficulty of detecting an engineered change in genomes that are highly variable from strain to strain. This problem will require comprehensive databases of whole genome sequences for more genetically variable biothreat bacteria and viruses. Preliminary work with microarrays for detecting synthetic elements or virulence genes and analytic bioinformatic approaches for whole genome sequence comparison to detect genetic engineering show promise for attacking this difficult problem but a large amount of future work remains.
Date: December 4, 2009
Creator: Slezak, T; Lenhoff, R; Allen, J; Borucki, M; Vitalis, E & Gardner, S
Partner: UNT Libraries Government Documents Department