David W. Ow

Bio: David W. Ow is an academic researcher from Chinese Academy of Sciences. The author has contributed to research in topics: Site-specific recombination & Recombinase. The author has an hindex of 45, co-authored 109 publications receiving 8269 citations. Previous affiliations of David W. Ow include Harvard University & University of California, San Diego.

Promises and Prospects of Phytoremediation

Abstract: We often think of plants primarily as a source of wood, food, and fiber Secondarily we may also appreciate their presence for aesthetic reasons as well as for "altruistically" providing habitat for other species Increasingly, however, their value as an environmental counterbalance to industrialization processes is being appreciated These processes include the burning of fossil fuels, generation of wastes (sewage, inorganic and organic solids, and effluents), and general water flow and processing Plants have long been recognized for their consumption of CO2 and, more recently, of other gaseous industrial byproducts (Simonich and Hites, 1994) Recently, their role in slowing the rate of global warming has been further appreciated in both the scientific and popular press Their use as a final water treatment and for disposal of sludge resulting from waste water treatment is centuries old (Hartman, 1975) The extensive literature concerning water and sludge treatment and the emerging field of air pollution abatement with plants will not be discussed here Instead, we focus on an emerging concept, "phytoremediation," the use of plants to remediate contamination of soil with organic or inorganic wastes Remediation of soil contamination by conventional engineering techniques often costs between $50 and $500 per ton Certain specialized techniques can exceed costs of $1000 per ton With an acre of soil (to a 3-foot depth) weighing approximately 4500 tons, this translates to a minimum cost of about a quarter million dollars per acre (Cunningham et al, 1995) It is not surprising that the cleanup of contaminated sites has not been proceeding at a rapid pace There is an active effort to develop new, more costeffective technologies to remediate contamination of such soils For the most part these efforts are being led by engineers and microbiologists More recently, however, green plant-based processes have begun receiving greater attention It has long been known that the life cycle of a plant has profound effects on the chemical, physical, and biological processes that occur in its immediate vicinity In the process of shoot and root growth, water and mineral acquisition, senescence, and eventual decay, plants can profoundly alter the surrounding soil The effects of many of these processes are apparent on the restoration of land at physically and chemically altered sites, ranging from road cuts to the site of the Mount St Helen's eruption These same plant-driven processes also occur in areas heavily impacted by industrial, mining, and urban activities One of the greatest forces driving increased emphasis on research in this area is the potential economic benefit of an agronomybased technology Growing a crop on an acre of land can be accomplished at a cost ranging from 2 to 4 orders of magnitude less than the current engineering cost of excavation and reburial There have been perhaps two dozen field tests to date; however, in many ways phytoremediation is still at its initial stages of research and development A comforting thought for plant biologists is that much of the research effort will be expected to center on a deeper understanding of basic plant processes So how do we envision phytoremediation working? The theory appears to be simple Agronomic techniques will be used to ready the contaminated soil for planting and to ameliorate chemical and physical limitations to plant growth Plants will then directly or indirectly absorb, sequester, and/or degrade the contaminant Plants and irrigation, fertilization, and cropping schemes will be managed to maximize this remedial effect By growing plants over a number of years, the aim is to either remove the pollutant from the contaminated matrix or to alter the chemical and physical nature of the contaminant within the soil so that it no longer presents a risk to human health and the environment As people who work in the remediation, herbicide development, and farming industries will attest, many weed species are remarkably tolerant of a wide range of organic and inorganic toxins Plants can thrive in soil contaminated to levels that are often orders of magnitude higher than current regulatory limits These limits are often set relatively independent of plant tolerance limits and are most often derived from human health and aquatic toxicology end points Ironically, many remediation plans begin with the destruction of the existing vegetation

Transient and Stable Expression of the Firefly Luciferase Gene in Plant Cells and Transgenic Plants

Abstract: The luciferase gene from the firefly, Photinus pyralis, was used as a reporter of gene expression by light production in transfected plant cells and transgenic plants. A complementary DNA clone of the firefly luciferase gene under the control of a plant virus promoter (cauliflower mosaic virus 35S RNA promoter) was introduced into plant protoplast cells (Daucus carota) by electroporation and into plants (Nicotiana tabacum) by use of the Agrobacterium tumefaciens tumor-inducing plasmid. Extracts from electroporated cells (24 hours after the introduction of DNA) and from transgenic plants produce light when mixed with the substrates luciferin and adenosine triphosphate. Light produced by the action of luciferase was also detected in undisrupted leaves or cells in culture from transgenic plants incubated in luciferin and in whole transgenic plants "watered" with luciferin. Although light was detected in most organs in intact, transgenic plants (leaves, stems, and roots), the pattern of luminescence appeared to reflect both the organ-specific distribution of luciferase and the pathway for uptake of luciferin through the vasculature of the plant.

Site‐specific integration of DNA into wild‐type and mutant lox sites placed in the plant genome

Abstract: The bacteriophage P1 Cre-lox site-specific recombination system has been used to integrate DNA specifically at lox sites previously placed in the tobacco genome. As integrated molecules flanked by wild-type lox sites can readily excise in the presence of Cre recombinase, screening for mutant lox sites that can resist excisional recombination was performed. In gene integration experiments, wild-type and mutant lox sites were used in conjunction with two strategies for abolishing post-integration Cre activity: (i) promoter displacement of a cre-expression construct present in the target genome; and (ii) transient expression of cre. When the promoter displacement strategy was used, integrant plants were recovered after transformation with constructs containing mutant lox sequences, but not with constructs containing wild-type lox sites. When cre was transiently expressed, integrant plants were obtained after transformation with either mutant or wild-type lox sites. DNA rearrangements at the target locus were less frequent when mutant lox sites were used. DNA integration at the genomic lox site was usually without additional insertions in the genome. Thus, the Cre-lox site-specific recombination system is useful for the single-copy integration of DNA into a chromosomal lox site.

Gene transfer with subsequent removal of the selection gene from the host genome.

Abstract: A general method of gene transfer that does not leave behind a selectable marker in the host genome is described. A luciferase gene was introduced into the tobacco genome by using the hygromycin phosphotransferase gene (hpt) as a linked selectable marker. Flanked by recombination sites from the bacteriophage P1 Cre/lox recombination system, the hpt gene was subsequently excised from the plant genome by the Cre recombinase. The Cre-catalyzed excision event in the plant genome was precise and conservative--i.e., without loss or alteration of nucleotides in the recombinant site. After removal of the Cre-encoding locus by genetic segregation, plants were obtained that had incorporated only the desired transgene. Gene transfer without the incorporation of antibiotic-resistance markers in the host genome should ease public concerns over the field release of transgenic organisms expressing such traits. Moreover, it would obviate the need for different selectable markers in subsequent rounds of gene transfer into the same host.

GUS fusions: beta‐glucuronidase as a sensitive and versatile gene fusion marker in higher plants.

Abstract: We have used the Escherichia coli beta-glucuronidase gene (GUS) as a gene fusion marker for analysis of gene expression in transformed plants. Higher plants tested lack intrinsic beta-glucuronidase activity, thus enhancing the sensitivity with which measurements can be made. We have constructed gene fusions using the cauliflower mosaic virus (CaMV) 35S promoter or the promoter from a gene encoding the small subunit of ribulose bisphosphate carboxylase (rbcS) to direct the expression of beta-glucuronidase in transformed plants. Expression of GUS can be measured accurately using fluorometric assays of very small amounts of transformed plant tissue. Plants expressing GUS are normal, healthy and fertile. GUS is very stable, and tissue extracts continue to show high levels of GUS activity after prolonged storage. Histochemical analysis has been used to demonstrate the localization of gene activity in cells and tissues of transformed plants.

Assaying chimeric genes in plants: The GUS gene fusion system

Abstract: DeJi~eitio, r Ge~e lrlt.~irm Much of tile attention and interest in modern molecular biology is fi~cussed on the regulation of gene expression. Factors influencing or mediating such regulation are often best studied using gene Alsions. Gene fusions can be defined its DNA constructions (perfi3rmed ill vitro or i~e Hvo) that result in the coding sequences from one gene (r@o,ter) being transcribed and/or translated under the direction of the controlling sequences of another gene (cmltrr Gene fusions can be of two general types, with many wtriatiuns within types. Transcriptional fusions are defined as fusions in which all protein coding sequences are derived from the reporter, with none from the cmm,//e~. Thus, although the m R N A produced may consist of sequences from both control/o and re/;o~ter, the protein synthesized will be encoded only by the reporter. Translational fusions, in contrast, are defined as those in which the polypeptide produced is the result of coding information provided by both copraoiler and reporter.

Heavy Metals in Contaminated Soils: A Review of Sources, Chemistry, Risks and Best Available Strategies for Remediation

Abstract: Scattered literature is harnessed to critically review the possible sources, chemistry, potential biohazards and best available remedial strategies for a number of heavy metals (lead, chromium, arsenic, zinc, cadmium, copper, mercury and nickel) commonly found in contaminated soils. The principles, advantages and disadvantages of immobilization, soil washing and phytoremediation techniques which are frequently listed among the best demonstrated available technologies for cleaning up heavy metal contaminated sites are presented. Remediation of heavy metal contaminated soils is necessary to reduce the associated risks, make the land resource available for agricultural production, enhance food security and scale down land tenure problems arising from changes in the land use pattern.

Cellular mechanisms for heavy metal detoxification and tolerance

Abstract: Heavy metals such as Cu and Zn are essential for normal plant growth, although elevated concentrations of both essential and non-essential metals can result in growth inhibition and toxicity symptoms. Plants possess a range of potential cellular mechanisms that may be involved in the detoxification of heavy metals and thus tolerance to metal stress. These include roles for the following: for mycorrhiza and for binding to cell wall and extracellular exudates; for reduced uptake or efflux pumping of metals at the plasma membrane; for chelation of metals in the cytosol by peptides such as phytochelatins; for the repair of stress-damaged proteins; and for the compartmentation of metals in the vacuole by tonoplast-located transporters. This review provides a broad overview of the evidence for an involvement of each mechanism in heavy metal detoxification and tolerance.

Methods for producing members of specific binding pairs

Abstract: A member of a specific binding pair (sbp) is identified by expressing DNA encoding a genetically diverse population of such sbp members in recombinant host cells in which the sbp members are displayed in functional form at the surface of a secreted recombinant genetic display package (rgdp) containing DNA encoding the sbp member or a polypeptide component thereof, by virtue of the sbp member or a polypeptide component thereof being expressed as a fusion with a capsid component of the rgdp. The displayed sbps may be selected by affinity with a complementary sbp member, and the DNA recovered from selected rgdps for expression of the selected sbp members. Antibody sbp members may be thus obtained, with the different chains thereof expressed, one fused to the capsid component and the other in free form for association with the fusion partner polypeptide. A phagemid may be used as an expression vector, with said capsid fusion helping to package the phagemid DNA. Using this method libraries of DNA encoding respective chains of such multimeric sbp members may be combined, thereby obtaining a much greater genetic diversity in the sbp members than could easily be obtained by conventional methods.