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Biomedical subjects

T C Hall

Publications and source records attributed to T C Hall.

At least 19 recordsLinked to original sources
PubMed2001

Kiddo, a new transposable element family closely associated with rice genes.

The promoter region of the rice ubiquitin2 (rubq2) gene was found to be polymorphic between japonica (T309) and indica (IR24) lines as the result of a 270-bp deletion in T309. A TTATA footprint in the T309 rubq2 promoter suggested that an excision event had occurred, and inspection of the 270-bp region present in IR24 revealed that it had all the characteristics of a miniature inverted repeat transposable element (MITE). Database searches showed that this element is a member of a new MITE family, which we have named Kiddo. Thirty-five complete Kiddo sequences were identified in existing rice genomic sequence databases. They could be arranged into four groups, within-group sequence identity was over 90%, with 65-75% identity between groups. The high sequence similarity within a group indicates that some Kiddo members were recently mobile and may still be active. An additional 24 decayed Kiddo sequences were detected. Interestingly, approximately 80% of 18 Kiddo members from annotated accessions lie within 530 bp of a coding sequence. That approximately 40% of Kiddo members present in genic regions reside in introns suggests that Kiddo transposition entails the use of both DNA and RNA intermediates, and may provide some insight into the origins of individual groups. DNA blot analysis showed that Kiddo is a rice-specific element, although one sequence with limited (72%) similarity to Kiddo group A was detected as a wheat EST. Kiddo family members may represent new molecular and phylogenetic markers, as well as representing valuable materials for studying the molecular mechanisms of MITE transposition.

Arabidopsis Proteins↗
PubMed2001

Chromatin structure and phaseolin gene regulation.

Chromatin structure, the organized packaging of DNA with histones in the nucleus, is now seen as a dynamic fabric that changes with development. Here, we use studies on the phaseolin (phas) gene that encodes a seed protein to show how chromatin structure interacts with the transcription machinery to accomplish rigorous spatial regulation of expression. In leaf and other vegetative tissues, a nucleosome is rotationally and translationally positioned over an ensemble of three phased TATA boxes, denying access to TBP. Current interest focuses on the mechanisms by which this architecture is remodeled during embryogenesis. The transcription factor PvALF is intrinsically involved, as are other non-histone proteins and abscisic acid. These concepts, and the possible modular nature of phas expression, are summarized together with speculations concerning the re-establishment of the nucleosome over the phas promoter during terminal stages of embryogenesis.

Chromatin↗
PubMed2001

The use of FISH in chromosomal localization of transgenes in rice.

Chromosomal location and local chromatin structure are thought to play important roles in the stability of transgene expression. Fluorescence in situ hybridization (FISH) is a cytogenetic technique that allows the localization of specific DNA sequences on chromosomes. It provides an excellent means to analyze the chromosomal environment of integrated transgenes, helping to assess the effect of position on gene expression. FISH analyses have been conducted on nuclear chromosomal DNA at metaphase, interphase, meiotic prophase (pachytene) and on extended chromatin fibers (DNA fiber-FISH) and naked DNA molecules. Despite the small size of rice chromosomes, FISH has been successfully accomplished to detect unique and repetitive DNA sequences. A detailed FISH procedure for the detection of small and single copy transgenes within the rice genome is described and the application of FISH to evaluate chromosomal location and the local chromatin structure of transgenes as parameters that could affect their expression is discussed.

Chromosome Mapping↗
PubMed2000

De novo activation of the beta-phaseolin promoter by phosphatase or protein synthesis inhibitors.

The promoter for the phaseolin (phas) bean seed protein gene adopts an inactive chromatin structure in leaves of transgenic tobacco. This repressive architecture, which confers stringent spatial regulation, is disrupted upon transcriptional activation during embryogenesis in a process that requires the presence of both a transcription factor (PvALF) and abscisic acid (ABA). Toward determining the need for de novo synthesis of proteins other than PvALF in transcriptional activation we explored the effect of several eukaryotic protein synthesis inhibitors. Surprisingly, cycloheximide (CHX), emetine, and verrucarin A were able to induce transcription from the phas promoter in tobacco and bean leaf tissue in the absence of either PvALF or ABA. This induction was decreased by the replication inhibitors hydroxyurea and aphidicolin but not by genistein or mimosine. Since protein phosphatases and kinases are essential components of the ABA signal transduction pathway, it is conceivable that CHX is also capable of inducing phosphorylation of proteins usually involved in ABA-mediated activation. Interestingly, okadaic acid, an inhibitor of serine/threonine phosphatase, also strongly activated transcription from the phas promoter. In contrast, the protein synthesis inhibitors anisomycin and puromycin did not activate transcription from the phas promoter, nor did the tyrosine phosphatase inhibitors phenylarsine oxide and sodium orthovanadate. These discrete but different results on transcriptional activation may reflect specific modes of action of the inhibitors, or they may reflect differential interactions of the inhibitors or of downstream events resulting from inhibitor activity with presently unknown components of the transcriptional activation system.

Cycloheximide↗
PubMed2000

Transgene silencing in monocots.

Plant gene silencing was originally thought to be a quirk of transformation procedures, but is now recognized to be a facet of vitally important gene regulatory systems, present in all organisms. Monocot plants, especially the grasses, play a foremost role in the agricultural economy of all nations, and their biotechnological manipulation offers great potential for both developed and developing countries. Here, we review reported instances of transgene silencing in monocots and relate the processes of transcriptional and post-transcriptional gene silencing (TGS, PTGS) in perspective to the rapidly burgeoning knowledge of these phenomena in many organisms. Recent findings include the involvement of an RNA-dependent RNA polymerase and a nuclease in PTGS systems and the close relationship between methylation and chromatin structure in TGS events.

Edible Grain↗
PubMed2000

Virus recovery is induced in Brome mosaic virus p2 transgenic plants showing synchronous complementation and RNA-2-specific silencing.

Nicotiana benthamiana plants expressing Brome mosaic virus (BMV) p2 protein complemented replication of RNAs1 + 3 but, surprisingly, supported little or no replication of RNA-2. Despite this, the p2 transgenic plants were able to support systemic migration of RNAs-1 and -3. Kinetic analyses showed identical degradation rates for RNAs-2 and -3, greatly detracting from the concept of an induction of an RNA-2-specific degradation system. Deletion analysis identified a 200-nucleotide sequence that may contribute to silencing in a context-specific manner. When R1 progeny of a severely silencing p2 transgenic line were tested for virus resistance, three different classes of reactions were observed. In class 1 and class 3 plants, the virus moved systemically and showed various extents of RNA-2 silencing. However, in class 2 plants, there was a stochastic onset of post-transcriptional silencing in the systemic leaves that was reminiscent of virus recovery. Plants showing recovery tended to have a greater number of transgene loci than did those exhibiting component-specific silencing. The induction of silencing did not appear to be dependent solely on the combined steady state levels of the transgene and viral RNA. Some plants transformed with a p2 frameshift construct showed a complete silencing phenotype, but none showed RNA-2-specific silencing. While the relationship between the two types of silencing remains unclear, we speculate that our observations reflect early events in the induction of virus recovery.

Bromovirus↗
PubMed1999

beta-Phaseolin gene activation is a two-step process: PvALF- facilitated chromatin modification followed by abscisic acid-mediated gene activation.

We have shown previously that a rotationally and translationally positioned nucleosome is responsible for the absence of transcriptional expression from the phaseolin (phas) gene promoter in leaf tissue and that the repressive chromatin structure is disrupted on transcriptional activation during embryogenesis. To investigate how the chromatin structure is modified, we ectopically expressed PvALF, a putative seed-specific phas activator, in leaf tissue of a tobacco line transgenic for a chimeric phas/uidA construct. DNase I footprinting in vivo revealed that the ectopic expression of PvALF resulted in remodeling of the chromatin architecture over the TATA region of the phas promoter but did not lead to transcriptional activation in the absence of abscisic acid (ABA). Treatment of the transgenic tobacco leaves with ABA in the absence of PvALF neither alleviated the repressive chromatin architecture nor activated transcription. However, in the presence of PvALF, high levels of beta-glucuronidase expression were obtained on exposure of leaves to ABA. These results reveal that expression from the phas promoter involves at least two discrete steps: chromatin potentiation by PvALF followed by ABA-mediated transcriptional activation.

Journal Article↗
PubMed1999

Organizational complexity of a rice transgene locus susceptible to methylation-based silencing.

Molecular analyses of a rice (Oryza sativa L.) transgene locus introduced using biolistic techniques revealed the presence of multiple copies of rearranged fragments, as well as an intact copy of the supplied constructs. Both the gene of interest (35S-Btt cryIIIA) and the selectable marker used (Ubi1-bar) were methylated and silenced. Additionally, vector sequences were present in great abundance and were also highly methylated, indicating that the entire transgene insert was marked for methylation. The rearrangement of input DNA resulted in interspersion of plasmid backbone regions with the gene of interest. Permutation of segments encoding the gene of interest and the selectable marker was also detected, perhaps explaining why sequences introduced on separate plasmids are frequently found to be inserted at the same locus. The 35S promoter contained several hotspots for fragmentation. These observations strongly support the concept that intrusive DNA is recognized by host surveillance systems and that transgene loci with anomalous structural organization are subjected to inactivation by processes such as methylation.

Bacillus thuringiensis↗
PubMed1998

Architectural specificity in chromatin structure at the TATA box in vivo: nucleosome displacement upon beta-phaseolin gene activation.

Extensive studies of the beta-phaseolin (phas) gene in transgenic tobacco have shown that it is highly active during seed embryogenesis but is completely silent in leaf and other vegetative tissues. In vivo footprinting revealed that the lack of even basal transcriptional activity in vegetative tissues is associated with the presence of a nucleosome that is rotationally positioned with base pair precision over three phased TATA boxes present in the phas promoter. Positioning is sequence-dependent because an identical rotational setting is obtained upon nucleosome reconstitution in vitro. A comparison of DNase I and dimethyl sulfate footprints in vivo and in vitro strongly suggests that this repressive chromatin architecture is remodeled concomitant with gene activation in the developing seed. This leads to the disruption of histone-mediated DNA wrapping and the assembly of the TATA boxes into a transcriptionally competent nucleoprotein complex.

Journal Article↗
PubMed1998

Longevity of 5-azacytidine-mediated gene expression and re-establishment of silencing in transgenic rice.

Epigenetic silencing of a bialaphos resistance (bar) gene in R1 progeny of a transgenic rice line was found to be meiotically stable since selfed (R2) progeny were also susceptible and the bar locus highly methylated. A high proportion of R2 seedlings germinated in the presence of 5-azacytidine (AzaC) were herbicide-resistant and also contained at least one unmethylated copy of the bar gene, further establishing the relationship between silencing and methylation. Restored bar gene expression was typically maintained for 20-50 days, but eventual methylation and silencing of the bar locus underscores the ability of the recipient genome to recognize and inactivate intrusive DNA.

Azacitidine↗
PubMed1997

The beta-phaseolin 5' matrix attachment region acts as an enhancer facilitator.

MARs found flanking the beta-phaseolin gene (phas) were tested for insulating activity in an enhancer blocking assay. True insulators should block enhancer dependent expression of a reporter gene when placed between the enhancer and a promoter. Insertion of phas 3' MAR or coding sequences lowered CaMV 35S enhancer driven GUS expression from the phas basal promoter, indicating a distance dependence of the 35S enhancer. 5' MAR or 5' MAR core fragments could not act as independent enhancers when fused to the phas basal promoter, and did not lower expression when inserted in the enhancer blocking assay construct, indicating that they facilitated 35S enhancer expression at a distance when located between the enhancer and the promoter.

Base Sequence↗
PubMed1997

Epigenetic transcriptional silencing and 5-azacytidine-mediated reactivation of a complex transgene in rice.

Despite a growing number of reports indicating non-Mendelian inheritance of transgene expression in monocots, no detailed description of the structure and stability of the transgene exists for transformants generated by direct DNA-transfer techniques, making the cause for these observations difficult to determine. In this paper we describe the complex organization of Btt cryIIIA and bar transgenes in rice (Oryza sativa L.) that displayed aberrant segregation in R1 progeny. Silencing rather than rearrangement of the bar gene was implicated because the herbicide-sensitive R1 plants had a DNA hybridization profile identical to that of the resistant R0 parent and R1 siblings. Genomic DNA analysis revealed substantial methylation of the Ubi1/bar sequences in silenced plants and, to a lesser degree, in herbicide-resistant plants, suggesting that the transgene locus was potentiated for silencing. Nuclease protection and nuclear run-on assays confirmed that silencing was due to transcriptional inactivation. Treatment of R2 progeny of silenced plants with 5-azacytidine resulted in demethylation of the Ubi1 promoter and reactivation of bar gene expression, demonstrating a functional relationship for methylation in gene silencing. These findings indicate that methylation-based silencing may be frequent in cereals transformed by direct DNA protocols that insert multiple, often rearranged sequences.

Azacitidine↗
PubMed1997

Paraneoplastic syndromes: mechanisms.

Neoplasms may affect distant host tissues, but they always involve normal physiologic mechanisms. Van R. Potter said that "Oncogeny is blocked ontogeny," so tumors are committed to their organ anlagen. Paraneoplastic mediators are appropriate to their blocked anlagen stages. Mediators may have autocrine, paracrine, or endocrine actions. However, their release and distribution depend on the mode of cell death, and the spatial relations to host vascularity. Invasiveness and metastasis are means of normal embryonic development, and not new cancer-specific properties. Nor do human cancers acquire new foreign genetic information; thus, they cannot express neoantigens nor be recognized by the host. However, tumors breach the blood-brain barrier and basement membrane separations of ectoderm from mesenchyme and release "forbidden" self-antigens, to which host immunocytes may respond causing cell- and antibody-mediated autoimmunity. This can damage normal host tissues by a "bystander" effect. Paraneoplastic mechanisms can also be analyzed as arising from three-way interactions between cells blocked in ontogeny, their molecular messengers, and the host tissues they target.

Apoptosis↗