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Biomedical subjects

W F Thompson

Publications and source records attributed to W F Thompson.

At least 55 records · Page 3Linked to original sources
PubMed1992

Both internal and external regulatory elements control expression of the pea Fed-1 gene in transgenic tobacco seedlings.

In previous studies using leaves of light-grown transgenic tobacco plants, we have shown that sequences located within the transcribed region of the pea Fed-1 gene (encoding ferredoxin I) are major cis-acting determinants of light-regulated mRNA accumulation. However, we show here that these internal sequences are less important for the Fed-1 light response in etiolated tobacco seedlings than they are in green leaves and that upstream elements confer organ specificity and contribute significantly to Fed-1 light responses in etiolated material. Light effects mediated by upstream response elements are thus most pronounced during the initial induction of gene activity, whereas internal elements play a more prominent role in modulating Fed-1 expression once the gene is already active.

Base Sequence↗
PubMed1991

Nuclear scaffolds and scaffold-attachment regions in higher plants.

DNA in the nuclei of eukaryotic organisms undergoes a hierarchy of folding to be packaged into interphase and metaphase chromosomes. The first level of packaging is the 11-nm nucleosome fiber, which is further coiled into a 30-nm fiber. Evidence from fungal and animal systems reveals the existence of higher order packaging consisting of loops of the 30-nm fibers attached to a proteinaceous nuclear scaffold by an interaction between the scaffold and specific DNA sequences called scaffold-attachment regions (SARs). Support for the ubiquitous nature of such higher order packaging of DNA is presented here by our work with plants. We have isolated scaffolds from tobacco nuclei using buffers containing lithium diiodosalicylate to remove histones and then using restriction enzymes to remove the DNA not closely associated with the scaffold. We have used Southern hybridization to show that the DNA remaining bound to the scaffolds after nuclease digestion includes SARs flanking three root-specific tobacco genes. This assay for SARs is termed the endogenous assay because it identifies genomic sequences as SARs by their endogenous association with the scaffold. Another assay, the exogenous assay, depends upon the ability of scaffolds to specifically bind exogenously added DNA fragments containing SARs. The tobacco scaffolds specifically bind a well-characterized yeast SAR, and cloned DNA fragments derived from the 3'-flanking regions of the root-specific genes are confirmed to contain SARs by this exogenous assay.

Journal Article↗
PubMed1991

High mobility group chromosomal proteins bind to AT-rich tracts flanking plant genes.

AT-rich sequences in the 5' flanking regions of several plant genes have been shown to bind nuclear proteins, but the nature of these proteins has remained largely unknown. We report here that certain plant high mobility group (HMG) chromosomal proteins can interact specifically (in the presence of excess non-specific competitor) with AT-rich sequences located upstream of the pea ferredoxin 1 gene (Fed-1) and a member of the wheat Em gene family. Binding was observed with highly purified preparations of HMGa or HMGb, but not with HMGc or HMGd. HMG-DNA complexes were similar to one of the two types of Fed-1 complexes we observed previously using pea nuclear extracts [7]. HMG binding to the Fed-1 DNA was localized to a region containing AT-rich sequences; very similar sequences are present 5' to Em and several other plants genes. Such sequences have been shown to bind unidentified nuclear proteins in a number of these systems. Binding experiments with a synthetic oligo (dA).oligo (dT) probe and competition experiments with synthetic DNA polymers suggest that HMG binding may depend upon structural features of AT-rich DNA rather than being sequence-specific. We discuss the implications of these findings and suggest a role for HMG binding which is consistent with previous evidence linking HMGs with transcriptionally competent chromatin.

Adenine↗
PubMed1991

Concatemer chain reaction: a Taq DNA polymerase-mediated mechanism for generating long tandemly repetitive DNA sequences.

The concatemer chain reaction (CCR) uses Taq DNA polymerase to synthesize double- or single-stranded DNA concatemers whose length and yield can be controlled by varying the number of thermal cycling steps. Although the reactions which occur in CCR are slower and more complex than in polymerase chain reaction (PCR), the practical application of the CCR technique is simple. The CCR technique is less expensive, faster, and easier than conventional methods for producing concatemers and gives greatly improved yields. The templates used in CCR may be: (i) double-stranded concatemer templates produced by ligation, (ii) double-stranded concatemers from previous CCRs, or (iii) single-stranded oligonucleotides consisting of one copy of the sense strand repeat and a complementary but overlapping repeat for the antisense strand. Different molar ratios and lengths (masses) of the two strands of the helix may be obtained. We have used both single-stranded and double-stranded concatemers as targets for RNA hybridization. Applications of this concatemer technology are discussed, including the use of concatemers as hybridization probes or targets in applications such as run-on transcription or analysis of repetitive DNA sequences.

Base Sequence↗
PubMed1989

The identification and localization of 33 pea chloroplast transcription initiation sites.

We have used a novel approach to produce a comprehensive transcription initiation map of the pea chloroplast genome. Sites were mapped by measuring the ability of DNA probes to protect 5' ends of transcripts that have been capped in vitro. Using this approach, at least 33 probes appear to contain one or more transcription start sites. A more precise location of some of these sites was obtained by hybrid selecting certain of these RNAs and determining their size both before and after RNase treatment. We have found at least one initiation site in front of every chloroplast gene cluster for which appropriate clones were available. In addition, we have found initiation sites within gene clusters previously shown to be co-transcribed. In one such case, we were able to locate a transcription start site for psbC within the coding sequence of psbD.

Base Sequence↗
PubMed1989

A developmentally regulated bud specific transcript in pea has sequence similarity to seed lectins.

We report a striking example of organ and stage specific gene expression in pea (Pisum sativum L.). We have identified a transcript to a previously isolated cDNA clone, pEA207 (WF Thompson et al. (1983) Planta 158: 487-500) which accumulates in the actively growing bud of the pea plant and is either absent or present at very low levels in the expanded leaves below the bud. The deduced amino acid sequence of pEA207 shows 49% similarity to the phytohemagglutinin lectin sequence of kidney bean (Phaseolus vulgaris) (LM Hoffman, DD Donaldson (1985) EMBO J 4: 883-889) and 37% similarity to that of the major pea seed lectin sequence (TJV Higgins et al. (1983) J Biol Chem 258: 9544-9549). It is also similar to seed lectins from five other legumes. All of the residues directly involved in metal binding by lectins are present in this sequence. We discuss the possibility that pEA207 encodes a sugar binding lectin-like polypeptide associated with the cell walls of actively growing plant cells.

Journal Article↗
PubMed1989

Characterization of a single copy gene encoding ferredoxin I from pea.

We have isolated, mapped, and sequenced a genomic clone containing the ferredoxin I (Fed-1) gene from Pisum sativum. The gene is present as a single copy per haploid genome. It has no introns, and it specifies a 753-nucleotide transcript encoding a 149-amino acid protein including a 52-residue transit peptide. Upstream sequences from Fed-1 contain several elements with similarity to transcriptional regulatory elements from RbcS and Cab genes, and gel mobility shift assays show that nuclear extracts from light-grown pea leaves contain one or more DNA binding activities specific for Fed-1 5'-flanking sequences. RbcS and Cab regulatory sequences are only weak competitors for this binding, however, and the RbcS and Cab similarities mostly lie outside of the region essential for binding. These data are discussed in terms of previously observed physiological differences between the light responses of Fed-1 and other genes.

Amino Acid Sequence↗
PubMed1988

Regulation of cytosine methylation in ribosomal DNA and nucleolus organizer expression in wheat.

Cytosine methylation has been studied in wheat rRNA genes at nucleolar organizers displaying different activities. The methylation pattern within a specific multigene locus is influenced by the number and type of rRNA genes in other rDNA loci in the cell. One CCGG site 164 base-pairs upstream from the start of transcription is preferentially unmethylated in some genes. Dominant, very active loci have a higher proportion of rRNA genes with unmethylated cytosine residues in comparison with recessive and inactive loci. It is concluded that cytosine methylation in rDNA is regulated and that the methylation pattern correlates with the transcription potential of an rRNA gene.

Binding Sites↗
PubMed1988

DNase I sensitivity of ribosomal RNA genes in chromatin and nucleolar dominance in wheat.

Ribosomal RNA genes at different nucleolar organizer (NOR) loci in hexaploid wheat are expressed at different levels. The degree of expression of a particular organizer depends on the genetic background, especially on the presence of other NOR loci. For example, when chromosome 1U of Aegilops umbellulata is introduced into the hexaploid wheat cultivar "Chinese Spring" the A. umbellulata NOR accounts for most of the nucleolar activity and seems to suppress the activity of the wheat NOR loci. Even in wild-type "Chinese Spring", the NOR on chromosome 1B is partially dominant to that on chromosome 6B, since the 1B locus is more active in spite of having fewer genes. We have previously shown that these and other examples of nucleolar dominance in wheat are associated with undermethylation of cytosine residues in certain regions of the dominant rDNA. Here, we show that rRNA genes at dominant loci are organized in a chromatin conformation that renders them more sensitive to DNase I digestion than other rRNA genes. In addition, we have mapped several DNase I-hypersensitive sites in the intergenic spacer region of the rDNA repeating unit. Some of these sites are located near the initiation region for the 45 S rRNA precursor, while others are associated with a series of short direct repeats 5' to the 45 S rRNA initiation site. The results are discussed in terms of a model in which repeated sequences in the wheat intergenic DNA are presumed to function as upstream promoters and transcriptional enhancers similar to those in Xenopus.

Binding Sites↗
PubMed1988

Phytochrome Regulation of Greening in Pisum: Chlorophyll Accumulation and Abundance of mRNA for the Light-Harvesting Chlorophyll a/b Binding Proteins.

A brief pulse of red light eliminates or reduces the lag in chlorophyll accumulation that occurs when dark-grown pea seedlings are transferred to continuous white light. The red light pulse also induces the accumulation of specific mRNAs. We compared time courses, escape from reversal by far-red light, and fluence-response behavior for induction of mRNA for the light-harvesting chlorophyll a/b binding proteins (Cab mRNA) with those for induction of rapid chlorophyll accumulation in seedlings of Pisum sativum cv Alaska. In both cases the time courses of low fluence and very low fluence responses diverged from each other in a similar fashion: the low fluence responses continued to increase for at least 24 hours, while the very low fluence responses reached saturation by 8 to 16 hours. Both responses escaped from reversibility by far-red slowly, approaching the red control level after 16 hours. The fluence-response curve for the Cab mRNA increase, on the other hand, showed threshold and saturation at fluences 10-fold lower than threshold and saturation values for the greening response. Therefore, the level of Cab mRNA, as measured by the presence of sequences hybridizing to a cDNA probe, does not limit the rate of chlorophyll accumulation after transfer of pea seedlings to white light. The Cab mRNA level in the buds of seedlings grown under continuous red light remained high even when the red fluence rate was too low to allow significant greening. In this case also, abundance of Cab mRNA cannot be what limits chlorophyll accumulation.

Journal Article↗
PubMed1988

Light effects on several chloroplast components in norflurazon-treated pea seedlings.

Changes occurring in several chloroplast components during Norflurazon-induced photobleaching of Pisum sativum seedlings were investigated. mRNA steady state levels of the chlorophyll a/b-binding protein of photosystem II, ferredoxin I, the small and large subunits of ribulose 1,5-bisphosphate carboxylase, and pEA214 and pEA207, two other light-responsive genes, were determined during chlorophyll photooxidation. Relative transcription rates were assayed in isolated nuclei. The results illustrate a complex set of interactions regulating expression of the nuclear and chloroplast genomes. Photobleaching was found to affect the expression of the various genes in different ways. While transcript levels of the chlorophyll a/b-binding protein decreased by more than 80% under photooxidative light conditions in carotenoid-deficient peas, levels of ferredoxin, the small and large subunits of ribulose 1,5-bisphosphate carboxylase, and pEA214 mRNAs were reduced by less than 50%. pEA207 mRNA levels, on the other hand, were resistant to the effects of photobleaching. Analyses of chlorophylls a and b and the chlorophyll a/b-binding protein suggest that accumulation of the protein and its mRNA are coordinated with chlorophyll abundance at several steps. In addition to post-transcriptional regulation at the level of mRNA and protein stability, there may exist coordination at the transcriptional stage.

Journal Article↗
PubMed1988

Nuclear-Cytoplasmic Partitioning of Phytochrome-Regulated Transcripts in Pisum sativum.

Nuclear and cytoplasmic mRNAs for several phytochrome-regulated genes were examined in Pisum seedlings in order to investigate possible light effects on mRNA partitioning between the nucleus and cytoplasm. Transcripts from each of five light-regulated genes exhibited different responses to a variety of light treatments, but for each transcript we observed a characteristic linear relationship between nuclear and cytoplasmic levels over a wide range of total transcript abundance. Different mRNAs are characterized by different nuclear-cytoplasmic ;partitioning coefficients', indicating that post-transcriptional events play a significant role in regulating the accumulation of these mRNAs during light induction.

Journal Article↗
PubMed1987

Developmental regulation of cytosine methylation in the nuclear ribosomal RNA genes of Pisum sativum.

Prominent features of the cytosine methylation pattern of the Pisum sativum nuclear ribosomal RNA genes have been defined. Cytosine methylation within the C-C-G-G sequence was studied using the restriction enzymes HpaII and MspI and gel blot hybridizations of the restriction digests. The extent to which particular features of the methylation pattern change during seedling development has also been determined. Total cellular DNA, purified from defined sections of pea seedlings grown under different lighting conditions, was analyzed with DNA hybridization probes derived from different portions of a cloned member of the nuclear rRNA gene family. By use of an indirect end-labeling technique, a map of 23 cleavable HpaII and/or MspI sites in genomic rDNA was constructed. The map covers about 90% of the rDNA repeat including the entire non-transcribed spacer region and most of the rRNA coding sequences. One notable feature of the map is that the most prominent HpaII site, located about 800 base-pairs upstream from the 5' end of the mature 18 S rRNA, is cleaved only in one of the two most abundant rDNA length variants (the short variant). With a gel blot assay specific for cleavage at this site, we estimated the HpaII sensitivity of DNA preparations from several stages of pea seedling development. We find that, while methylation is generally low in young seedlings, DNA obtained from the apical buds of pea seedlings is highly methylated. Further, the methylation level of rDNA within the pea bud decreases as the buds are allowed to develop under continuous white light. Our data, taken together with published studies on pea seedling development, indicate that cytosine methylation levels may be related to the regulated expression of the nuclear rRNA genes in pea.

Cell Nucleus↗
PubMed1987

Light-regulated changes in DNase I hypersensitive sites in the rRNA genes of Pisum sativum.

We have examined the rDNA chromatin of Pisum sativum plants grown with or without exposure to light for the presence of DNase I hypersensitive sites and possible developmental changes in their distribution. Isolated nuclei from pea seedlings were incubated with various concentrations of DNase I. To visualize the hypersensitive sites, DNA purified from these nuclei was restricted and analyzed by gel blot hybridization. We find that several sites exist in both the coding and noncoding regions of rDNA repeating units. Several of the sites in the nontranscribed spacer region are present in the light but are absent in the dark. Conversely, the hypersensitive sites within the mature rRNA coding regions are present in the dark but absent in the light. There are two major length variants of the rRNA genes in P. sativum var. Alaska. The sites in the nontranscribed spacer region that appear during the light treatment occur only in the shorter of these two length variants in this cultivar.

Journal Article↗
PubMed1987

Specific mRNA and rRNA Levels in Greening Pea Leaves during Recovery from Iron Stress.

Hydroponically grown pea seedlings (Pisum sativum L., cv Alaska) were subjected to Fe stress for 10 to 16 days to produce mature chlorotic leaves. Greening was initiated by adding Fe to the nutrient solution. The levels of chlorophylls, chloroplast, and cytoplasmic rRNAs, and specific chloroplast- and nucleus-encoded mRNAs were all significantly lower in leaves developing during iron stress than in nonstressed leaves. In plants greening after addition of Fe, nuclear transcripts encoding chlorophyll a/b-binding protein and the small subunit of ribulose bisphosphate carboxylase/oxygenase increased about 5-fold in abundance following an 18 to 24 hour lag, as did the chloroplast-encoded transcript for the large subunit of the carboxylase/oxygenase. Chloroplast rRNA showed an increase over that in continually stressed control leaves only after a 40 hour lag. The chloroplast-encoded transcript encoding the Q(B)-binding 32 kilodalton polypeptide of Photosystem II showed little change during greening. Chlorophyll itself increased gradually after a lag period of 24 hours, with an increase in chlorophyll a slightly preceding that of chlorophyll b. Kinetic considerations suggest that the changes observed represent a coordinate series of events initiated by readdition of Fe and occurring in parallel. Though accumulation of mRNA for light-harvesting, chlorophyll-a/b-binding protein might limit chlorophyll accumulation at the onset, subsequent changes in the mRNA do not parallel chlorophyll changes. All three of the mRNAs showing recovery on addition of Fe to Fe-stressed plants undergo sharp diurnal fluctuations in abundance. Such fluctuations are comparable to those in nonstressed controls (mRNA for light-harvesting protein) or considerably more pronounced (mRNAs for carboxylase large and small subunits). The carboxylase small subunit mRNA and that for light-harvesting chlorophyll-binding protein were measured under constant conditions of light and temperature. Though a rhythm in greening leaves was hard to detect, it was prominent in the Fe-sufficient controls, persisting undamped through three full cycles for both mRNAs, and hence is probably circadian.

Journal Article↗
PubMed1986

The watermelon mitochondrial URF-1 gene: evidence for a complex structure.

We have cloned and sequenced a fragment of watermelon mitochondrial DNA (mtDNA) which contains a gene homologous to mitochondrial URF-1 (Unidentified Reading Frame-1) of vertebrates, Drosophila yakuba and Aspergillus nidulans. URF-1 is thought to encode a component of the respiratory chain NADH dehydrogenase. Two coding regions in the watermelon gene are separated by approximately 1,450 bp of untranslatable DNA. These two exons encode the central portions of URF-1, and are highly conserved. We postulate that three additional exons, selected by their map location and amino acid homology to other URF-1 sequences, encode the remainder of the polypeptide. This is the first description of a plant mitochondrial gene with multiple introns.

Amino Acid Sequence↗