PubMed Health⌕ Search

Biomedical subjects

M Bevan

Publications and source records attributed to M Bevan.

At least 37 records · Page 2Linked to original sources
PubMed1994

A flower-specific Myb protein activates transcription of phenylpropanoid biosynthetic genes.

Synthesis of flavonoid pigments in flowers requires the co-ordinated expression of genes encoding enzymes in th phenylpropanoid biosynthetic pathway. Some cis-elements involved in the transcriptional control of these genes have been defined. We report binding of petal-specific activities from tobacco and Antirrhinum majus (snapdragon) to an element conserved in promoters of phenylpropanoid biosynthetic genes and implicated in expression in flowers. These binding activities were inhibited by antibodies raised against Myb305, a flower-specific Myb protein previously cloned from Antirrhinum by sequence homology. Myb305 bound to the same element and formed a DNA-protein complex with the same mobility as the Antirrhinum petal protein in electrophoretic mobility shift experiments. Myb305 activated expression from its binding site in yeast and in tobacco protoplasts. In protoplasts, activation also required a G-box-like element, suggesting co-operation with other elements and factors. The results strongly suggest a role for Myb305-related proteins in the activation of phenylpropanoid biosynthetic genes in flowers. This is consistent with the genetically demonstrated role of plant Myb proteins in the regulation of genes involved in flavonoid synthesis.

Base Sequence↗
PubMed1994

The projections from the parafascicular thalamic nucleus to the subthalamic nucleus and the striatum arise from separate neuronal populations: a comparison with the corticostriatal and corticosubthalamic efferents in a retrograde fluorescent double-labelling study.

The parafascicular thalamic nucleus projects to the subthalamic nucleus and the striatum. Double-retrograde fluorescent tracing was used to determine whether these projections arise from the same neurons via axon collaterals. True Blue was injected into the subthalamic nucleus and Nuclear Yellow was injected into the striatum of each rat and the parafascicular thalamic nucleus was examined under the fluorescence light-microscope. Individual parafascicular neurons were not double-labelled with the tracers. The True Blue- and Nuclear Yellow-labelled neurons wee located in different parts of the parafascicular nucleus ipsilateral to the injections. In the rostral part of the parafascicular nucleus, True Blue-labelled neurons were located ventral to the fasciculus retroflexus, and in the caudal part of the nucleus. True Blue-labelled neurons were located close to the medial and lateral borders of fasciculus retroflexus. Nuclear Yellow-labelled neurons were found mainly to encircle the fasciculus retroflexus in the rostral part of the parafascicular nucleus and in the dorsolateral sector of the caudal part of the parafascicular nucleus. Double-labelled neurons were, however, found in the cortex. The proportion of neurons projecting to both the subthalamic nucleus and the striatum accounted for 38% of the total number of cortiscosubthalamic neurons in the prefrontal cortex, 15.5% in the cingulate cortex and 9% in the sensorimotor cortex. The present finding of an individualization between the parafascicular efferents to the subthalamic nucleus and the striatum emphasize the importance of this projection and provides further evidence of the associative functions attributable to the subthalamic nucleus.

Animals↗
PubMed1994

Separate cis sequences and trans factors direct metabolic and developmental regulation of a potato tuber storage protein gene.

The expression of genes encoding patatin, a major tuber protein, is highly tissue-specific but is also modulated by exogenous sucrose. The patterns of transcription observed in potato plants could be due to mechanisms conferring tuber-specificity or they could reflect the concentrations of sucrose found in different tissues. To distinguish between these possibilities, a detailed examination was made of the function of a region of the promoter previously implicated in conferring tissue-specific and sucrose-inducible expression. Internal deletions of this region revealed three separate functional domains regulating expression. The B repeat region acted as a positive activator of transcription in the tuber and was also responsible for a degree of sucrose-inducibility. The distal region of the A repeat repressed transcription in leaf and tuber tissue, while the proximal region of the A repeat was able to confer sucrose-responsiveness. Each of these regions specifically bound nuclear proteins which may be putative transcription factors involved in conferring these responses. The region found to confer sucrose-inducible expression was conserved among some other genes that are also regulated by exogenous sucrose.

Base Sequence↗
PubMed1993

Transcriptional control of plant storage protein genes.

The accumulation of plant storage proteins is controlled primarily by the transcriptional activation of their genes. Two classes of storage proteins, the zygotic or seed-specific, and the somatic, such as tuber proteins, have been studied. Gene expression analysis in transgenic plants has defined small regions of the promoters of such genes that are able to confer the appropriate patterns of expression. Protein-DNA interactions, both in vivo and in vitro, have revealed proteins that bind to regions implicated in expression, and these may be transcription factors. Promoter deletion analysis has determined the role of some of these DNA-binding proteins, such as in determining tissue-specificity or levels of expression. A common theme linking the expression of both classes of storage proteins is the involvement of metabolite levels in directly controlling gene expression.

Base Sequence↗
PubMed1993

Transcription of two members of a gene family encoding phenylalanine ammonia-lyase leads to remarkably different cell specificities and induction patterns.

Phenylalanine ammonia-lyase (PAL) catalyses the first committed step in the biosynthesis of phenylpropanoids, which perform a variety of functions in plant development and in their interactions with the environment. French bean contains a small family of genes encoding PAL and two of these genes, PAL2 and PAL3, have been shown to be differentially expressed at the mRNA level in bean tissues. The transcriptional activities of the PAL2 and PAL3 genes have been investigated by fusing their promoters to the reporter gene beta-glucuronidase (GUS) and transforming these constructs into Arabidopsis, potato and tobacco. The PAL2- and PAL3-GUS constructs exhibited different spatial and temporal patterns of expression during development and in response to environmental stimuli. The consistency of these data with previous mRNA analysis in bean suggests that the differential expression of these two PAL genes is, at least in part, a function of their promoter activities. New patterns of PAL2 and PAL3 promoter activities were also characterized. Some species-specific differences in GUS expression were observed and these may reflect differences in phenylpropanoid metabolism or the signals that modulate PAL gene transcription.

Arabidopsis↗
PubMed1992

Control of gene expression in tobacco cells using a bacterial operator-repressor system.

We have investigated the efficacy of using the Escherichia coli lac operator-repressor system to control plant gene expression. The lacI gene was modified to allow optimal expression in plant cells and then placed downstream of the cauliflower mosaic virus (CaMV) 35S RNA promoter. This construct was introduced into tobacco plants by leaf disc transformation. Transgenic tobacco plants synthesized significant quantities of LacI protein (up to 0.06% of total soluble protein). We have used the E.coli beta-glucuronidase gene (gus) as the reporter gene by placing it downstream of the maize chlorophyll a/b binding protein (CAB) gene promoter. Lac operators were introduced into several positions within the CAB promoter and operator-free plasmid was used as control. Repression was assessed by comparing the transient expression from CAB-operator-gus reporter constructs in protoplasts expressing lac protein, with that in control cells not expressing the repressor. Repression varied between 10 and 90% with different operator positions. Transient assays were also performed in the presence of the inducer, isopropyl-beta-D-thiogalactoside (IPTG). In lacI protoplasts the presence of IPTG manifested itself in a 4.2-fold relief of repression. The study was extended to show regulation of expression in stable transformants. Tobacco transformants harbouring a CAB-operator-gus reporter construct and the lacI gene were shown to have repressed GUS levels, but in the presence of IPTG, repression was relieved 15-fold. We conclude that the lac repressor can enter the plant cell nucleus, find its cognate operator sequence in the chromatin to form a repressor--operator complex and effectively block transcription of a downstream gene.

Bacterial Proteins↗
PubMed1992

DNA-binding properties of cloned TATA-binding protein from potato tubers.

A full-length cDNA clone encoding the TATA-binding protein (TBP), the DNA-binding component of the general transcription factor TFIID was cloned from potato tubers. The DNA sequence of this cDNA indicated that the predicted potato protein was very similar to cloned TBP from other species. Genomic southern analysis showed that TBP is encoded in the potato genome as a low-copy-number sequence. The potato TBP cDNA clone was shown to encode a functional protein that interacts in a sequence-specific way with the promoter region of a class-1 potato patatin gene. Functional analysis of carboxy-terminal truncated derivatives of potato TBP showed that important components of DNA binding were located within the carboxy-terminal 54 amino acids. Kinetic and thermodynamic properties of in vitro synthesised potato TBP were investigated, and demonstrated strict salt and temperature preferences for maximum DNA binding activity. In addition on and off-rate measurements showed that both association and dissociation of TBP from DNA is slow. The specific and the non-specific equilibrium constants Ks and Kn were calculated as 5 x 10(9) M-1 and 3.65 x 10(4) M-1 respectively. These results indicate that the interaction of potato TBP with the patatin promoter is highly specific.

Amino Acid Sequence↗
PubMed1992

Autocatalytic activity of the tobacco etch virus NIa proteinase in viral and foreign protein sequences.

The small nuclear inclusion (NIa) protein of the tobacco etch virus (TEV) is synthesized initially as part of a genome-derived high M(r) precursor. The NIa protein releases itself from this genome-derived precursor by self-cleavage, or an autocatalytic processing event. Cleavage between specific glutamine-glycine dipeptides at the N and C termini generates the 430 amino acid or 49,000 M(r) (49K) NIa protein. The requirements of this autocatalytic release, or cis cleavage, were examined by constructing gene cassettes encoding the TEV NIa protein which could be ligated into particular locations in cDNA of the TEV genome and also into foreign gene DNA sequences. Using cell-free transcription and translation systems, polyproteins containing TEV NIa sequences were synthesized and assayed for (i) autocatalysis and (ii) the ability of a functional NIa proteinase, purified from plant tissue, to cleave in bimolecular or trans reactions various artificial polyproteins which contained an inactive form of the NIa proteinase. The NIa self-cleavage events required an active proteinase sequence and a consensus TEV cleavage site sequence at the N and C termini. These results were consistent for NIa protein sequences placed at a foreign TEV cleavage site or in unrelated proteins. Differences were noted in the trans cleavage of these sites.

Amino Acid Sequence↗
PubMed1991

pH sensitive liposomes provide an efficient means of sensitizing target cells to class I restricted CTL recognition of a soluble protein.

Exogenous antigens are normally endocytosed and enter the class II pathway of processing and presentation. It had been shown earlier that soluble antigen could be introduced into the class I pathway of processing and presentation by osmotic loading. In this report, we have demonstrated that OVA containing liposomes that destabilize on exposure to low pH, referred to as pH sensitive liposomes, could sensitize target cells to lysis by class I MHC-restricted OVA-specific CTL. However, OVA-containing pH insensitive liposomes, native OVA, or OVA subjected to the same protocol as was used to make the liposomes, failed to sensitize targets to OVA-specific CTL lysis. The pH sensitive liposomal approach was less toxic and more efficient (about 20-fold) in delivering than the osmotic loading approach. The pH liposome approach may prove valuable to study CTL recognition characteristics of less available proteins such as viral proteins.

Animals↗
PubMed1991

New patterns of gene activity in plants detected using an Agrobacterium vector.

A vector has been designed that contains a truncated CaMV (cauliflower mosaic virus) 35S promoter fused to a receptor gene encoding beta-glucuronidase (GUS), placed adjacent to the left border sequence of an Agrobacterium vector. In potato plants transformed with this vector, different patterns of transcription were detected at high frequency using in situ assays for GUS activity. Previous studies in Drosophila using analogous vectors have shown that the new patterns of transcription in many cases reflect the patterns of expression of genes adjacent to the site of vector insertion. If this is also the case in plants, the vector described here will be useful in identifying the activity of genes in different cell types and will assist in determining their function.

Blotting, Southern↗
PubMed1991

Ethylene and a Wound Signal Modulate Local and Systemic Transcription of win2 Genes in Transgenic Potato Plants.

The transcriptional regulation of a win2-beta-glucuronidase gene fusion in transgenic potato (Solanum tuberosum) plants by wounding and ethylene has been analyzed. In common with other genes that are expressed in response to mechanical or chemical stress, win2 is transcribed at the site of injury and also in distant undamaged parts of the wounded plant. Similar kinetics of induction and patterns of transcription were observed in response to mechanical, wounding, elicitor, or arachidonic acid application. Experiments involving the use of chemicals that inhibited ethylene action, and those that increased ethylene production, showed that local induction of win2 transcription did not have an absolute requirement for ethylene, but ethylene was necessary for high levels of expression. In contrast, systemic expression of win2 required both a putative wound signal and ethylene. Ethylene alone had no direct effect on win2 gene expression in the absence of wounding.

Journal Article↗
PubMed1990

Transcriptional regulation of a patatin-1 gene in potato.

Patatin is an abundant glycoprotein in the tubers of potato plants that has a lipid acyl hydrolase activity. Fusions of the promoter of patatin genes that are highly expressed in tubers with the reporter gene encoding beta-glucuronidase (GUS) have shown that patatin transcription has a high degree of tuber specificity. Patatin transcription was also inducible in other organs of transgenic potato by growth on high concentrations of sucrose. Experiments were conducted to define regions of the patatin promoter that confered tuber specific expression and sucrose inducibility. Sequences between -40 and -400 bp and between -400 and -957 bp of the transcriptional start site were able to confer tuber-specific expression on a heterologous truncated promoter. The cell specificity of GUS transcription in the transformants indicated that organ specificity was possibly determined by source-sink relationships of sucrose, or a metabolite of sucrose, in the whole plant.

Base Sequence↗
PubMed1989

Tissue- and cell-specific activity of a phenylalanine ammonia-lyase promoter in transgenic plants.

Phenylalanine ammonia-lyase (PAL) catalyses the first step in the biosynthesis of phenylpropanoids, which form a wide variety of plant secondary products. The transcription of PAL is regulated in response to various factors that induce the accumulation of flavonoids, lignin and compounds thought to be involved in plant defence reactions. The 5' upstream sequence of a PAL gene from Phaseolus vulgaris was fused to the coding region of the reporter gene encoding beta-glucuronidase (GUS), and transformed into potato and tobacco plants. Histochemical analysis of GUS expression showed that the PAL promoter was active in specific cell types that accumulated phenylpropanoid derivatives in response to mechanical wounding, and also during normal development of the xylem and flower. In xylem that had undergone secondary thickening, GUS activity occurred in rays of cells thought to be the xylem parenchyma. It was postulated that PAL activity in these cells could provide intermediates for lignin synthesis in xylem vessels that had terminally differentiated.

Ammonia-Lyases↗
PubMed1989

Differential expression within a family of novel wound-induced genes in potato.

Wounding in higher plants leads to an increased synthesis of specific messenger RNAs. A cDNA clone complementary to a wound-induced message from potato tubers was used to isolate a lambda clone from a genomic library of Solanum tuberosum var. Maris Piper. DNA sequence analysis has shown that this single genomic clone contains two novel wound-induced genes, called win1 and win2, organised in close tandem array. The coding sequences of these two genes are highly homologous and are interrupted by a single intron. However, the sequences of the introns and flanking regions have diverged widely. Win1 and win2 encode cysteine-rich proteins of 200 and 211 amino-acids, respectively, which show striking homologies to several chitin-binding proteins. Southern analysis of genomic DNA has shown that win1 and win2 are members of a small multi-gene family which is estimated to have a minimum of five members per haploid genome of Maris Piper and appears to be conserved within the Solanaceae. We have shown by Northern analysis and S1 mapping that the two genes exhibit differential organ-specific expression after the wounding of a potato plant.

Amino Acid Sequence↗
PubMed1989

The function of plant heat shock promoter elements in the regulated expression of chimaeric genes in transgenic tobacco.

A series of deletion mutants of a soybean heat shock (hs) gene promoter was generated and linked to the chloramphenicol acetyl transferase (CAT) coding sequence. These chimaeric promoter/reporter gene constructs were introduced into tobacco and thermoregulated expression of CAT activity was examined in leaf extracts. Three different types of gene fusions were tested using two different BIN19 vector constructions: (1) translational fusion between the N-terminus of the protein coding sequence of the heat shock gene Gmhsp17.3-B and CAT; (2) transcriptional fusions between the 5' nontranslated RNA regions of Gmhsp17.3-B and CAT; and (3) promoter fusions joining the hs promoter upstream sequences to the TATA box sequence of the delta CaMV 35S-CATter vector. Alternatively, multiple copies of a synthetic deoxyoligonucleotide with the soybean hs consensus element (HSE2) were used. Heat inducible CAT activities were detected except in plants containing a transcriptional fusion devoid of all but 18 nucleotides at the 5' terminus of the hs gene transcript. CAT activity was detectable in these plants only during the recovery at 25 degrees C after a hs (40 degrees C). Overlapping HSE-like promoter sequences seem to be necessary for the induction of heat inducible transcription of linked genes; synthetic HSE2 sequences have the capacity to reconstitute a hs promoter in combination with a TATA box sequence. Effective translation during hs seems to require sequences in the 5' non-translated leader of the hs protein mRNA; these sequences can be functionally replaced by the 5' leader sequence of the delta CaMV 35S promoter.

Amino Acid Sequence↗
PubMed1989

Failure of aspartame to affect seizure susceptibility in kindled rats.

The effect of aspartame administered by gavage to rats on amygdala and hippocampal kindled seizures was assessed. Despite the administration of a wide range of doses (25-2000 mg/kg) no evidence for an effect of aspartame on afterdischarge threshold or seizure strength was obtained when testing was done at a time when serum and brain levels of neutral amino acids are known to be significantly elevated as a result of this treatment. There is controversy whether dietary aspartame (N-L-aspartyl-L-phenylalanine 1-methyl ester), a food additive sweetner, can lead to seizures in susceptible humans and in laboratory animals. A proseizure effect of high consumption of aspartame has been alleged (Wurtman, 1985; Walton, 1986) and denied (Gaull, 1985). Recent studies using mice have yielded mixed results. Thus, Kim and Kim (1986) and Pinto and Maher (1988) observed potentiating effects of high loads of aspartame on chemically induced seizures, but Nevins, Arnolde and Haigler (1986) observed no effect on chemical and ECS seizures. We used the electrical kindling model of epilepsy to assess whether aspartame can alter seizure threshold or strength in rats. The kindled response is highly repeatable and stable and has been shown to be sensitive to a large variety of pharmacological treatments (Racine, 1978) and to dietary manipulation (McCann, Cain and Philbrick, 1983).

Amygdala↗
PubMed1987

Functional analysis of sequences required for transcriptional activation of a soybean heat shock gene in transgenic tobacco plants.

The 5' DNA sequences involved in the thermal inducibility of the soybean heat shock gene hs6871 were analysed in transgenic tobacco plants. The transcriptional activity of various in vitro generated deletion mutants was examined by Northern blot analysis, S1 nuclease mapping and dot-blot hybridization. At least 181 bp upstream from the translational start site are sufficient for thermal induction at 40 degrees C and correct initiation of transcripts. Full promoter activity with the induction of wild-type levels of transcripts requires additional upstream sequences contained within 439 bp 5' to the coding sequence. Our results suggest that faithful regulation and the generation of high levels of hs6871-specific mRNA depend on the presence of sequences which show homology to the 14-bp heat shock consensus element of Drosophila and, in addition, on as yet unidentified enhancer-like upstream sequences.

Journal Article↗