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Biomedical subjects

A L Paul

Publications and source records attributed to A L Paul.

17 recordsLinked to original sources
PubMed2001

The fungicidal and phytotoxic properties of benomyl and PPM in supplemented agar media supporting transgenic arabidopsis plants for a Space Shuttle flight experiment.

Fungal contamination is a significant problem in the use of sucrose-enriched agar-based media for plant culture, especially in closed habitats such as the Space Shuttle. While a variety of fungicides are commercially available, not all are equal in their effectiveness in inhibiting fungal contamination. In addition, fungicide effectiveness must be weighed against its phytotoxicity and in this case, its influence on transgene expression. In a series of experiments designed to optimize media composition for a recent shuttle mission, the fungicide benomyl and the biocide "Plant Preservative Mixture" (PPM) were evaluated for effectiveness in controlling three common fungal contaminants, as well as their impact on the growth and development of arabidopsis seedlings. Benomyl proved to be an effective inhibitor of all three contaminants in concentrations as low as 2 ppm (parts per million) within the agar medium, and no evidence of phytotoxicity was observed until concentrations exceeded 20 ppm. The biocide mix PPM was effective as a fungicide only at concentrations that had deleterious effects on arabidopsis seedlings. As a result of these findings, a concentration of 3 ppm benomyl was used in the media for experiment PGIM-01 which flew on shuttle Columbia mission STS-93 in July 1999.

Agar↗
PubMed2001

Transgene expression patterns indicate that spaceflight affects stress signal perception and transduction in arabidopsis.

The use of plants as integral components of life support systems remains a cornerstone of strategies for long-term human habitation of space and extraterrestrial colonization. Spaceflight experiments over the past few decades have refined the hardware required to grow plants in low-earth orbit and have illuminated fundamental issues regarding spaceflight effects on plant growth and development. Potential incipient hypoxia, resulting from the lack of convection-driven gas movement, has emerged as a possible major impact of microgravity. We developed transgenic Arabidopsis containing the alcohol dehydrogenase (Adh) gene promoter linked to the beta-glucuronidase (GUS) reporter gene to address specifically the possibility that spaceflight induces the plant hypoxia response and to assess whether any spaceflight response was similar to control terrestrial hypoxia-induced gene expression patterns. The staining patterns resulting from a 5-d mission on the orbiter Columbia during mission STS-93 indicate that the Adh/GUS reporter gene was activated in roots during the flight. However, the patterns of expression were not identical to terrestrial control inductions. Moreover, although terrestrial hypoxia induces Adh/GUS expression in the shoot apex, no apex staining was observed in the spaceflight plants. This indicates that either the normal hypoxia response signaling is impaired in spaceflight or that spaceflight inappropriately induces Adh/GUS activity for reasons other than hypoxia.

Alcohol Dehydrogenase↗
PubMed1999

Higher-order chromatin structure: looping long molecules.

Chromatin structure plays a variety of roles in eukaryotes, ranging from the structural organization of the genome to the facilitation of transcription factors and remodeling of individual gene promoters. Higher-order chromatin structure typically refers to those structural features of the genome that serve to facilitate large-scale condensation and packaging. It is becoming increasingly clear, however, that large-scale features that create loop domains play an important role in the management and functional organization of the genome as well. Recently, plant models have made significant contributions to our understanding of higher-order chromatin structures in eukaryotes.

Animals↗
PubMed1998

Permeabilized Arabidopsis protoplasts provide new insight into the chromatin structure of plant alcohol dehydrogenase genes.

New data from permeabilized protoplasts have expanded our view of the 5'DNase I hypersensitive area of the Arabidopsis Adh gene derived from nuclei. DNase I hypersensitivity analyses conducted with permeabilized protoplasts from Arabidopsis cell cultures indicates that there are four distinct sites of hypersensitivity centered around positions -425, -325, -200, and -60. The hypersensitive site at -200 coincides with an in vitro hypersensitive site created by purified transcription factors bound to a G-box element. The G-box is a functional cis element that plays a role in the signal transduction of hypoxia and other stresses in Adh. The data presented in this paper support the notion that G-box-related elements may also play a role in defining chromatin structure. The new Arabidopsis data are discussed within the context of what is known about the chromatin structures and regulation of two other plant Adh genes; maize Adh1 and Adh2. The chromatin of the maize Adh1 promoter is divided into a region that is constitutively hypersensitive to DNase I (-700 to -160) and an inducibly hypersensitive region (-140 and -40). There are several sequence elements within the hypersensitive regions bound by proteins in vivo. The anaerobic response element is the most well characterized and functions in the detection of hypoxia. The maize Adh2 gene promoter is constitutively hypersensitive to DNase I, with the exception of a small region that extends to include the TATA box as the gene becomes active. Several cis elements in the Adh2 promoter are bound by factors in vivo. One, at -160, is a functional element that acts as an activator in vascular tissue. The overall goal of our research with the Adh genes from maize and Arabidopsis is to gain further insight into the relationships between the regulation of gene transcription and chromatin structure in plants as it is clear that all the necessary components that characterize regulated gene activity may not be found simply by elucidating the linear sequence of nucleotides that lie 5' to the protein coding regions and finding proteins capable of binding the promoter in vitro.

Alcohol Dehydrogenase↗
PubMed1998

Higher order chromatin structures in maize and Arabidopsis.

We are investigating the nature of plant genome domain organization by using DNase I- and topoisomerase II-mediated cleavage to produce domains reflecting higher order chromatin structures. Limited digestion of nuclei with DNase I results in the conversion of the >800 kb genomic DNA to an accumulation of fragments that represents a collection of individual domains of the genome created by preferential cleavage at super-hypersensitive regions. The median size of these fragments is approximately 45 kb in maize and approximately 25 kb in Arabidopsis. Hybridization analyses with specific gene probes revealed that individual genes occupy discrete domains within the distribution created by DNase I. The maize alcohol dehydrogenase Adh1 gene occupies a domain of 90 kb, and the maize general regulatory factor GRF1 gene occupies a domain of 100 kb in length. Arabidopsis Adh was found within two distinct domains of 8.3 and 6.1 kb, whereas an Arabidopsis GRF gene occupies a single domain of 27 kb. The domains created by topoisomerase II-mediated cleavage are identical in size to those created by DNase I. These results imply that the genome is not packaged by means of a random gathering of the genome into domains of indiscriminate length but rather that the genome is gathered into specific domains and that a gene consistently occupies a discrete physical section of the genome. Our proposed model is that these large organizational domains represent the fundamental structural loop domains created by attachment of chromatin to the nuclear matrix at loop basements. These loop domains may be distinct from the domains created by the matrix attachment regions that typically flank smaller, often functionally distinct sections of the genome.

Alcohol Dehydrogenase↗
PubMed1997

Localization of 14-3-3 proteins in the nuclei of arabidopsis and maize.

It has been demonstrated that 14-3-3 proteins are present in the nuclei of Arabidopsis thaliana and Zea mays cells using laser scanning confocal microscopy and immunocytochemistry with monoclonal antibodies against plant 14-3-3 proteins. Confirmation of nuclear localization provides insight into the range of functions normally attributed to 14-3-3 proteins, especially since the association of 14-3-3s with transcription factors is (thus far) a phenomenon unique to plants, and since 14-3-3 proteins do not possess a recognizable nuclear targeting sequence.

14-3-3 Proteins↗
PubMed1996

Transcription factor veracity: is GBF3 responsible for ABA-regulated expression of Arabidopsis Adh?

Assignment of particular transcription factors to specific roles in promoter elements can be problematic, especially in systems such as the G-box, where multiple factors of overlapping specificity exist. In the Arabidopsis alcohol dehydrogenase (Adh) promoter, the G-box regulates expression in response to cold and dehydration, presumably through the action of abscisic acid (ABA), and is bound by a nuclear protein complex in vivo during expression in cell cultures. In this report, we test the conventional wisdom of biochemical approaches used to identify DNA binding proteins and assess their specific interactions by using the G-box and a nearby half G-box element of the Arabidopsis Adh promoter as a model system. Typical in vitro assays demonstrated specific interaction of G-box factor 3 (GBF3) with both the G-box and the half G-box element. Dimethyl sulfate footprint analysis confirmed that the in vitro binding signature of GBF3 essentially matches the footprint signature detected in vivo at the G-box. Because RNA gel blot data indicated that GBF3 is itself induced by ABA, we might have concluded that GBF3 is indeed the GBF responsible in cell cultures for binding to the Adh G-box and is therefore responsible for ABA-regulated expression of Adh. Potential limitations of this conclusion are exposed by the fact that other GBFs bind the G-box with the same signature as GBF3, and subtle differences between in vivo and in vitro footprint signatures indicate that factors other than or in addition to GBF3 interact with the half G-box element.

Abscisic Acid↗
PubMed1994

Expression of active, processed ricin in transgenic tobacco.

The cDNA encoding the plant toxin precursor preproricin was introduced into tobacco via Agrobacterium tumefaciens-mediated gene transfer. Transgenic plants were assayed for type II ribosome-inactivating protein expression and activity. Western blot analysis of soluble leaf extracts using anti-ricin a-chain (RTA) antibodies identified 34- and 32-kDa proteins, which were electrophoretically indistinguishable from castor seed RTA. Analysis with anti-ricin b-chain (RTB) antibodies identified both a 34-kDa protein major band, which co-migrated with castor seed RTB, and a 30-kDa protein minor band. Enzyme-linked immunoassay of the transgenic leaf extracts with anti-RTA and anti-RTB indicated microgram per gram production on a fresh weight basis of soluble extractable recombinant ricin. Sugar binding enzyme-linked immunoassay employing an immobilized glycoprotein, asialofetuin, and anti-RTB antibodies confirmed the characteristic type II ribosome-inactivating protein galactose binding lectin activity of the recombinant ricin. The enzymatic activity of recombinant ricin was characterized for cell-free translation inhibition, as well as for overall cytotoxicity. A 50% inhibitory dose of 3 x 10(-11) M was observed for the immunoreactive leaf extract material using a rabbit reticulocyte translation inhibition assay, while a 50% lethal dose of 1 x 10(-12) M was calculated with human T-lymphotropic virus-1 infected leukemic T-cells.

Agrobacterium tumefaciens↗
PubMed1994

In vivo footprinting identifies an activating element of the maize Adh2 promoter specific for root and vascular tissues.

In vivo footprinting identifies four putative cis elements of Adh2 that interact with protein factors within the DNase I hypersensitive domains of the 5' flanking region. The power of in vivo footprinting to identify functionally significant sites within a gene promoter was tested by biochemical and transgenic analyses of the putative element at position -160. Biochemical analyses show that proteins isolated from maize cell suspensions will bind to the Adh2 promoter in vitro to generate a footprint at -160 identical to that seen in vivo. The partially purified factor bound to the promoter in vitro can be specifically competed with fragments of DNA containing the element sequence, further demonstrating that a specific protein generates the footprint over that sequence. Transgenic analyses indicate that the -160 element is a functional element of the maize Adh2 promoter that acts as an activator in the meristem and vascular tissue of roots and in the vascular tissue of stems and leaves.

Alcohol Dehydrogenase↗
PubMed1993

Osmium tetroxide footprinting of a scaffold attachment region in the maize Adh1 promoter.

Osmium tetroxide (OsO4) reacts with the thymine residues of double-stranded DNA, but thymines that are unpaired or under torsional stress are hyperreactive. Although OsO4 hyperreactivity has been primarily utilized to identify Z-DNA structures in supercoiled plasmids, OsO4 will also identify other torsional perturbations of DNA. In this study, OsO4 was used to footprint an AT-rich region (between -780 and -500) of the maize Adh1 promoter. Hyperreactive sites were identified both in vitro and in vivo in an area that coincides with AT motifs similar to those found in scaffold attachment regions. Further, the region of OsO4 hyperreactivity lies within a fragment of DNA that is associated with the nuclear scaffold in histone-depleted nuclei.

Alcohol Dehydrogenase↗
PubMed1992

Chemical detection of Z-DNA within the maize Adh1 promoter.

Z-DNA is a left-handed helix which can form within tracts of alternating purines and pyrimidines. Tracts of potential Z-DNA identified by sequence inspection are often noted within regulatory portions of genes, but evidence that these tracts of sequence actually exist as Z-DNA is very limited, and not available for any plant gene. In this study, the chemical probes osmium tetroxide, diethylpyrocarbonate and hydroxylamine were used to show that a tract of alternating purines and pyrimidines in the Adh1 promoter (from -311 to -325) actually assumes a Z-DNA conformation under superhelical stress in vitro.

Alcohol Dehydrogenase↗
PubMed1991

In vivo footprinting reveals unique cis-elements and different modes of hypoxic induction in maize Adh1 and Adh2.

The transcriptional activation of maize alcohol dehydrogenase-1 (Adh1) and alcohol dehydrogenase-2 (Adh2) is accompanied by changes in the chromatin structure within the 5'-flanking region of each gene. The positions of DNA-binding factors bound to the 5'-flanking regions were determined by in vivo dimethyl sulfate footprinting of maize suspension cultures over 8 hours of hypoxic induction. In Adh1 there are two types of DNA-binding factors associated with the promoter region. One set of factors is constitutively associated with the cis-regulatory anaerobic response element, whereas two additional factors bind only after Adh1 has been induced by hypoxic stress. Returning hypoxically stressed cells to an aerobic environment restores the dimethyl sulfate footprint observed for the uninduced Adh1 gene. In contrast, all of the factors bound to the 5'-flanking region of Adh2 are constitutively present and unchanged by hypoxia. There is one footprint site common to both Adh1 and Adh2, but it is not an anaerobic response-like element.

Aerobiosis↗
PubMed1990

In vivo and in vitro characterization of protein interactions with the dyad G-box of the Arabidopsis Adh gene.

Expression of the alcohol dehydrogenase (Adh) and ribulose-1,5-bisphosphate carboxylase small subunit (RbcS) genes of higher plants is cell-type-specific and environmentally inducible. However, the tissues in which these two genes are expressed, their modes of induction, and their protein functions are quite distinct. Adh is expressed in non-green tissue, induced by anaerobiosis, and repressed in leaves. RbcS is only expressed in green tissue. An 8-base pair G-box element (5'-CCACGTGG-3') is associated with light-induced expression of RbcS and chalcone synthase. The same sequence is also present in the 5'-flanking region of Arabidopsis thaliana Adh, and this sequence is associated with a trans-acting factor in vivo. We report here that in vitro Adh G-box binding activity is present in crude whole cell extracts of both cell culture and leaves of Arabidopsis. The authenticity of in vitro Adh G-box binding is supported by in vivo and in vitro dimethylsulfate footprinting. A clear in vivo Adh G-box footprint occurs in cell cultures, but comparable in vivo binding to the Adh G-box does not occur in leaves. Therefore, there does not appear to be a direct correlation between the presence of the G-box factor in a tissue and its binding to the Adh G-box.

Alcohol Dehydrogenase↗
PubMed1987

Constitutive and anaerobically induced DNase-I-hypersensitive sites in the 5' region of the maize Adh1 gene.

DNase-I-hypersensitive sites have been characterized in a plant gene, maize Adh1 (which encodes alcohol dehydrogenase 1). It has been generally recognized in animal genes that the chromatin of the 5' flanking region can be characterized by the accessibility of its DNA to the nuclease DNase I (EC 3.1.21.1), indicating which areas in the promoter are "open" to nuclear factors. The 5' region of the maize Adh1 gene contains two distinct DNase-I-hypersensitive regions, one constitutively present from position -160 to -700 and one that is anaerobically induced from position -35 to -150. The constitutive region contains three major hypersensitive sites, one of which corresponds in part to a region of potential Z-DNA. The induced hypersensitive region includes TATAA at -38 and CAAT at -100 as well as other potential regulatory sequences.

Journal Article↗
PubMed1986

Regulation of genes encoding the large subunit of ribulose-1,5-bisphosphate carboxylase and the photosystem II polypeptides D-1 and D-2 during the cell cycle of Chlamydomonas reinhardtii.

Synthesis of the major chloroplast proteins is temporally regulated in light-dark-synchronized Chlamydomonas cells. We have used cloned chloroplast DNA probes, and in vitro and in vivo protein synthesis to examine the cell cycle regulation of photosystem II polypeptides D-1 and D-2, and the large subunit of ribulose-1,5-bisphosphate carboxylase (RuBPCase LS). Synthesis and accumulation of D-1 and D-2 mRNAs occurs during the first half of the light period (G1), correlating with increasing synthesis of the polypeptides. Rifampicin, added immediately before the light period, inhibited the normal increase in D-1, D-2 polypeptide synthesis. During the dark period D-1, D-2 mRNAs persist at high levels despite reduced rates of mRNA synthesis and translation during this period. Cell-free translation analyses indicate that the D-1 mRNA present during the dark period is efficient at directing synthesis of the D-1 precursor in vitro. We conclude that expression of the psbA (D-1) and psbD (D-2) genes are regulated primarily at the transcriptional level during the light-induction period but at the translational level for the remainder of the cell cycle. Transcripts of the RuBPCase LS gene (rbcL) are also found at high levels during the light and dark periods but, unlike D-1 and D-2, LS mRNA levels do not increase until the last half of the light period and measurable synthesis and accumulation of this mRNA occurs during the dark. Furthermore, induction of LS polypeptide synthesis during the light period is insensitive to rifampicin. We conclude that LS production is regulated primarily at the translational level during the cell cycle.

Cell Cycle↗