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Biomedical subjects

D G Barker

Publications and source records attributed to D G Barker.

At least 19 recordsLinked to original sources
PubMed2003

Efficient transformation of Medicago truncatula cv. Jemalong using the hypervirulent Agrobacterium tumefaciens strain AGL1.

The efficiency of Agrobacterium tumefaciens transformation of the model legume Medicago truncatula cv. Jemalong (genotype 2HA) was evaluated for strains LBA 4404, C58pMP90, C58pGV2260 and AGL1. Binary vectors carrying promoter- gus/ gfp reporter gene fusions and the nptII gene as selectable marker were used for plant in vitro transformation/regeneration. The highest transformation efficiency was obtained with the disarmed hypervirulent strain AGL1 (Ti plasmid TiBo542), for which the percentage of explants forming kanamycin (Km)-resistant calli was double that obtained with each of the other three strains. In addition, we were able to reduce the time necessary for plant regeneration using AGL1, with 24% of the explants generating Km-resistant transgenic plantlets within only 4-5 months of culture. Transgene expression in planta was analysed and found to be conserved in the T(1) descendents.

Agrobacterium tumefaciens↗
PubMed2001

Agrobacterium rhizogenes-transformed roots of Medicago truncatula for the study of nitrogen-fixing and endomycorrhizal symbiotic associations.

Medicago truncatula, a diploid autogamous legume, is currently being developed as a model plant for the study of root endosymbiotic associations, including nodulation and mycorrhizal colonization. An important requirement for such a plant is the possibility of rapidly introducing and analyzing chimeric gene constructs in root tissues. For this reason, we developed and optimized a convenient protocol for Agrobacterium rhizogenes-mediated transformation of M. truncatula. This unusual protocol, which involves the inoculation of sectioned seedling radicles, results in rapid and efficient hairy root organogenesis and the subsequent development of vigorous "composite plants." In addition, we found that kanamycin can be used to select for the cotransformation of hairy roots directly with gene constructs of interest. M. truncatula composite plant hairy roots have a similar morphology to normal roots and can be nodulated successfully by their nitrogen-fixing symbiotic partner, Sinorhizobium meliloti. Furthermore, spatiotemporal expression of the Nod factor-responsive reporter pMtENOD11-gusA in hairy root epidermal tissues is indistinguishable from that observed in Agrobacterium tumefaciens-transformed lines. M. truncatula hairy root explants can be propagated in vitro, and we demonstrate that these clonal lines can be colonized by endomycorrhizal fungi such as Glomus intraradices with the formation of arbuscules within cortical cells. Our results suggest that M. truncatula hairy roots represent a particularly attractive system with which to study endosymbiotic associations in transgenically modified roots.

Fabaceae↗
PubMed2001

Medicago truncatula ENOD11: a novel RPRP-encoding early nodulin gene expressed during mycorrhization in arbuscule-containing cells.

Leguminous plants establish endosymbiotic associations with both rhizobia (nitrogen fixation) and arbuscular mycorrhizal fungi (phosphate uptake). These associations involve controlled entry of the soil microsymbiont into the root and the coordinated differentiation of the respective partners to generate the appropriate exchange interfaces. As part of a study to evaluate analogies at the molecular level between these two plant-microbe interactions, we focused on genes from Medicago truncatula encoding putative cell wall repetitive proline-rich proteins (RPRPs) expressed during the early stages of root nodulation. Here we report that a novel RPRP-encoding gene, MtENOD11, is transcribed during preinfection and infection stages of nodulation in root and nodule tissues. By means of reverse transcription-polymerase chain reaction and a promoter-reporter gene strategy, we demonstrate that this gene is also expressed during root colonization by endomycorrhizal fungi in inner cortical cells containing recently formed arbuscules. In contrast, no activation of MtENOD11 is observed during root colonization by a nonsymbiotic, biotrophic Rhizoctonia fungal species. Analysis of transgenic Medicago spp. plants expressing pMtENOD11-gusA also revealed that this gene is transcribed in a variety of nonsymbiotic specialized cell types in the root, shoot, and developing seed, either sharing high secretion/metabolite exchange activity or subject to regulated modifications in cell shape. The potential role of early nodulins with atypical RPRP structures such as ENOD11 and ENOD12 in symbiotic and nonsymbiotic cellular contexts is discussed.

Amino Acid Sequence↗
PubMed1998

An Arg/Lys-rich core peptide mimics TRBP binding to the HIV-1 TAR RNA upper-stem/loop.

TRBP is a cellular protein that binds to the HIV-1 leader RNA, TAR. Circular dichroism experiments have shown that a 24 amino acid peptide (TR1), located within a dsRNA binding domain (dsRBD) of TRBP, binds TAR with a 3:1 stoichiometry, eliciting a conformational change involving base unstacking. The binding characteristics of synthetic structural variants of TAR indicate that guanine residues play a key role in the TR1-RNA interaction and that binding sites exist in the upper-stem/loop and lower stem region of TAR. Deletion analysis of TR1 has led to the identification of a 15 amino acid subpeptide (TR13) which is necessary and sufficient to bind to the high affinity upper-stem/loop binding site of TAR. Alanine scanning of TR13 has revealed that mutations in either Lys or Arg residues result in altered TAR-binding, and molecular modelling/docking experiments have shown that the two Arg residues of TR13 can interact with two appropriately spaced guanine residues in the upper-stem/loop of TAR. The TR13 lysine residues appear to be essential for maintaining structural integrity and the correct positioning of the Arg side-chains. We propose that TRBP binds TAR by means of a "2-G hook" motif and that the binding specificity of this particular member of the family of double-stranded RNA-binding proteins lies within the highly conserved dsRBD core motif. Finally, our results also suggest that TRBP may function in vivo by modifying the tertiary structure of TAR RNA.

Amino Acid Sequence↗
PubMed1998

MtENOD16 and 20 are members of a family of phytocyanin-related early nodulins.

We have identified two single-copy genes from the model legume. Medicago truncatula (MtENOD16 and 20) whose expression can be correlated with early stages of root nodulation and whose predicted coding sequences are partially homologous to both pea/vetch ENOD5 and soybean N315/ENOD55. Database searching and sequence alignment have defined the encoded early nodulins as a distinct sub-family of phytocyanin-related proteins, although the absence of key ligands implies that they are unlikely to bind copper. Molecular modelling based on known phytocyanin structure has been used to predict the 3-dimensional conformation of the principle globular domain of MtENOD16/20. Additional structural features common to both early nodulin and phytocyanin precursors include an N-terminal transit peptide, a highly variable (hydroxy)proline-rich sequence which probably undergoes extensive post-translational modification, and a hydrophobic C-terminal tail.

Amino Acid Sequence↗
PubMed1994

Rhizobium meliloti Nod factors elicit cell-specific transcription of the ENOD12 gene in transgenic alfalfa.

Extracellular lipo-oligosaccharides of Rhizobium, known as Nod factors, play a key role in the molecular signal exchange which leads to the specific nitrogen-fixing symbiotic association between the soil microbe and its host legume. The biological activity of Nod factors and their perception by the host plant during the earliest stages of the Rhizobium/legume interaction have been studied using transgenic alfalfa carrying a fusion between the promoter of the early nodulin gene MtENOD12 and the beta-glucuronidase (GUS) reporter gene. Histochemical staining has shown that GUS accumulates specifically in the differentiating root epidermis, prior to and during root hair emergence, within 2-3 h following the addition of purified Rhizobium meliloti Nod factors. This precocious transcriptional activation of the MtENOD12 gene, reminiscent of that observed after inoculation with intact Rhizobium, implies that the Nod factor signal can be perceived at a developmental stage preceding root hair formation. GUS activity can be detected following treatment with a wide range of R. meliloti Nod factor concentrations down to 10(-13) M, and furthermore, this rapid response to the bacterial elicitor appears to be non-systemic. Significantly, MtENOD12-GUS expression is not observed after inoculation with a R. meliloti nodH mutant which synthesizes exclusively non-sulphated Nod factors. Indeed purified Nod factors which lack the sulphate substituent are approximately 1000-fold less active than their sulphated counterparts. Thus, the triggering of ENOD12 transcription in the alfalfa root epidermis is a rapid molecular response which is subject to the same host-specificity determinant (Nod factor sulphation) that governs the interaction between alfalfa and its bacterial symbiont.

Carbohydrate Sequence↗
PubMed1993

Differential expression within the glutamine synthetase gene family of the model legume Medicago truncatula.

The glutamine synthetase (GS) gene family of Medicago truncatula Gaertn. contains three genes related to cytosolic GS (MtGSa, MtGSb, and MtGSc), although one of these (MtGSc) appears not to be expressed. Sequence analysis suggests that the genes are more highly conserved interspecifically rather than intraspecifically: MtGSa and MtGSb are more similar to their homologs in Medicago sativa and Pisum sativum than to each other. Studies in which gene-specific probes are used show that both MtGSa and MtGSb are induced during symbiotic root nodule development, although not coordinately. MtGSa is the most highly expressed GS gene in nodules but is also expressed to lower extents in a variety of other organs. MtGSb shows higher levels of expression in roots and the photosynthetic cotyledons of seedlings than in nodules or other organs. In roots, both genes are expressed in the absence of an exogenous nitrogen source. However the addition of nitrate leads to a short-term, 2- to 3-fold increase in the abundance of both mRNAs, and the addition of ammonium leads to a 2-fold increase in MtGSb mRNA. The nitrogen supply, therefore, influences the expression of the two genes in roots, but it is clearly not the major effector of their expression. In the discussion section, the expression of the GS gene family of the model legume M. truncatula is compared to those of other leguminous plants.

Ammonia↗
PubMed1992

Lectin genes from the legume Medicago truncatula.

We report the cloning and characterization of two lectin genes from Medicago truncatula, designated Mtlec1 and Mtlec2. The two genes show a high degree of homology and apparently belong to a small multigene family. Mtlec1 appears to encode a functional lectin with 277 amino acids, whereas Mtlec2 is probably non-functional, since a frameshift mutation (insertion of two nucleotides) leads to premature translation termination after only 98 amino acids. The deduced amino acid sequence of the polypeptide MtLEC1 suggests that this lectin is a metalloprotein with Glc/Man specificity.

Amino Acid Sequence↗
PubMed1992

Rhizobium meliloti elicits transient expression of the early nodulin gene ENOD12 in the differentiating root epidermis of transgenic alfalfa.

To study the molecular responses of the host legume during early stages of the symbiotic interaction with Rhizobium, we have cloned and characterized the infection-related early nodulin gene MtENOD12 from Medicago truncatula. In situ hybridization experiments have shown that, within the indeterminate Medicago nodule, transcription of the MtENOD12 gene begins in cell layers of meristematic origin that lie ahead of the infection zone, suggesting that these cells are undergoing preparation for bacterial infection. Histochemical analysis of transgenic alfalfa plants that express an MtENOD12 promoter-beta-glucuronidase gene fusion has confirmed this result and further revealed that MtENOD12 gene transcription occurs as early as 3 to 6 hr following inoculation with R. meliloti in a zone of differentiating root epidermal cells which lies close to the growing root tip. It is likely that this transient, nodulation (nod) gene-dependent activation of the ENOD12 gene also corresponds to the preparation of the plant for bacterial infection. We anticipate that this extremely precocious response to Rhizobium will provide a valuable molecular marker for studying early signal exchange between the two symbiotic organisms.

Amino Acid Sequence↗
PubMed1991

Synchronous expression of leghaemoglobin genes in Medicago truncatula during nitrogen-fixing root nodule development and response to exogenously supplied nitrate.

Two leghaemoglobin genes from the diploid, autogamous Medicago truncatula (Mtlb1 and Mtlb2) have been cloned and their nucleotide sequences determined. The deduced amino acid sequences encoded by these two genes differ significantly (18%), confirming that they belong to different sub-groups of Medicago leghaemoglobin genes. RNAse protection experiments have been used to show that both genes are transcriptionally active, and are expressed specifically in the nitrogen-fixing root nodule of M. truncatula. Whilst Mtlb1 mRNA is present at approximatively 3-fold higher steady-state levels than Mtlb2 mRNA, the transcription of both genes is triggered concomitantly during nodule development (5 days after inoculation with Rhizobium meliloti), and the ratio of the steady-state levels of the two mRNA species remains constant throughout nodule maturation. When the growth medium of nodulated M. truncatula is supplemented with 5 mM KNO3 over a period of 2-3 days there is a progressive drop in specific nitrogen fixation activity to only 20-25% of the original level. This is accompanied with a parallel and synchronous reduction in the quantities of mRNA corresponding to both Mtlb1 and Mtlb2. By contrast, the expression of the nodule parenchyma-specific gene ENOD2 is not significantly modified following nitrate treatment, clearly demonstrating differences in tissue-specific gene regulation in response to combined nitrogen.

Amino Acid Sequence↗
PubMed1987

Molecular characterisation of the DNA ligase gene, CDC17, from the fission yeast Schizosaccharomyces pombe.

We have sequenced a 4200-base-pair fragment of Schizosaccharomyces pombe DNA which encompasses the entire DNA ligase gene, CDC17. S1 mapping has enabled us to identify two small introns (40 and 62 nucleotides) at the 5' end of the coding region of the gene and their 3' internal conserved sequences match the CTRAY consensus found in other S. pombe introns. The major transcription initiation and 3' polyadenylation sites have been mapped and are preceded by higher eukaryotic-like TATA and AATAAA sequences respectively. Furthermore, the CDC17 mRNA carries a poly(A) tail whose length (approximately 250 nucleotides) is typical of that found in higher eukaryotic mRNAs, and is in contrast to the much shorter polyadenylated sequences found for the mRNAs of the budding yeast, Saccharomyces cerevisiae. The deduced amino acid sequence of the S. pombe DNA ligase predicts a protein of 86182 daltons, and an overall 53% homology with the same enzyme from S. cerevisiae. In particular, a stretch of 24 amino acids with 100% sequence homology spans the putative ATP-binding region which is also conserved in T4 and T7 bacteriophage DNA ligases.

Base Composition↗
PubMed1987

Characterisation of an autonomously replicating sequence from the fission yeast Schizosaccharomyces pombe.

A DNA sequence has been isolated from Schizosaccharomyces pombe which promotes high frequency transformation of plasmids in the same organism. It is closely linked to the DNA ligase gene CDC17 and has therefore been named ARS17 although in structure it differs substantially from ARS elements in Saccharomyces cerevisiae. ARS17 spans some 1.8 kb of DNA and deletion of any part of this region affects activity. Moreover, there does not appear to be any short sequence which is, by itself, sufficient for high frequency transformation. ARS17 lies between and partly overlaps two divergently transcribed genes and it is extremely AT rich. It lacks the consensus sequence found in S. cerevisiae ARSs and it has no ARS activity in S. cerevisiae.

Base Sequence↗
PubMed1987

The CDC8 transcript is cell cycle regulated in yeast and is expressed coordinately with CDC9 and CDC21 at a point preceding histone transcription.

Using cultures synchronized by elutriator size selection or a feed-starve protocol, we have shown that the CDC8 gene is periodically expressed in the Saccharomyces cerevisiae cell cycle. The transcript level increases some 30-fold in late G1, reaching a peak at approximately the G1/S phase boundary. The timing of this event was compared with those of CDC9 and CDC21, which are already known to be periodically transcribed, and all three genes were found to be expressed at the same time in the cell cycle. In contrast, the histone H2A gene appeared to be expressed distinctly later in the cell cycle than these three genes and this was further investigated by examining expression of all four genes in a cdc4 mutant, held at the restrictive temperature. CDC8, CDC9, and CDC21 were once again expressed together and a complete fluctuation in levels occurred, whereas the histone gene was not expressed, presumably because the cdc4 block point precedes the point of histone expression. The three CDC genes may therefore be coordinately controlled, while the histone gene is regulated separately.

Cell Cycle↗
PubMed1986

Expression of polyoma virus middle-T antigen in Saccharomyces cerevisiae.

The polyoma middle-T gene, lacking its intron, was inserted into a yeast expression plasmid containing the phosphoglycerate kinase promoter. Such plasmids transformed yeast at low frequency and these transformants expressed middle-T antigen at a level of approximately 0.1% cell protein. Furthermore, expression of this protein was frequently lost during growth in liquid culture and this loss of middle-T was accompanied by a twofold increase in the rate of growth. The spontaneous production of a truncated middle-T antigen, lacking the C terminus, was also observed; the expression of this protein did not inhibit the growth rate of the cells. Recovery and analysis of the expression plasmids encoding the truncated molecule showed that a single C X G base pair had been deleted from a run of nine consecutive C X G base pairs (Pyr nucleotide 1239--1247) within the middle-T coding region. This frame-shift mutation results in premature termination of the protein and loss of the strongly hydrophobic region of the molecule believed to be responsible for the membrane association of middle-T antigen.

Antigens, Polyomavirus Transforming↗
PubMed1986

Periodic transcription as a means of regulating gene expression during the cell cycle: contrasting modes of expression of DNA ligase genes in budding and fission yeast.

Using cultures synchronised by three independent procedures, we have shown that the CDC9 gene, coding for DNA ligase, is periodically expressed in the Saccharomyces cerevisiae cell cycle. The level of CDC9 transcript increases many fold in late G1 reaching a peak at about the G1/S phase boundary and preceding the peak in histone message by some 20 min. The level of DNA ligase itself also fluctuates, showing the expected pattern for a stable enzyme synthesised periodically. In contrast, the transcript from the DNA ligase gene (CDC17) of Schizosaccharomyces pombe is present at a constant level throughout the cell cycle, and no fluctuation in amount was detected, although the histone H2A showed the expected periodic synthesis. Furthermore, DNA ligase activity remains at a constant level during the S. pombe cell cycle showing that there is unlikely to be any form of translational control. These contrasting modes of expression of the DNA ligase genes in the two organisms suggests that when periodic transcription is observed from an essential cell cycle gene, it may have no particular significance for regulating progress through the cell cycle. Also, regulatory circuits may be less well conserved between organisms than the processes they control and thus different organisms may utilise quite different modes of control to achieve the same ends.

Cell Cycle↗
PubMed1986

Induction of yeast DNA ligase genes in exponential and stationary phase cultures in response to DNA damaging agents.

UV-irradiation of stationary phase cells of Saccharomyces cerevisiae and Schizosaccharomyces pombe leads to a 9-fold and 90-fold increase in transcript levels from the respective DNA ligase genes CDC9 and CDC17, whereas exponential cells show only 3-fold and 2-fold increases. Induction of CDC9 after MMS treatment and gamma-irradiation was also observed by using a CDC9-lacZ translational fusion and assaying for beta-galactosidase. Surprisingly, irradiation of S. cerevisiae induces only a 50% increase in DNA ligase itself, probably reflecting the extremely high in vivo stability of the enzyme. The UV-induction of ligase may be part of a "fail-safe" mechanism which, together with the enzyme stability, ensures adequate supplies of this essential enzyme.

DNA Damage↗
PubMed1986

The expression in meiosis of genes which are transcribed periodically in the mitotic cell cycle of budding yeast.

The mitotic cell cycle genes CDC 8, 9 and 21 in Saccharomyces cerevisiae, together with the histone H2A gene, are transcribed discontinuously in meiosis. Message from all four genes initially declines in amount, then increases abruptly to reach maximal levels during premeiotic DNA synthesis before again declining. This response occurs only in meiotic cells; in asporogenous diploids the transcript simply declines in amount. In contrast, message from four genes with no known specific meiotic function (including the actin gene) shows the same profile in both sporogenous and asporogenous diploids. In mitotic cells the three CDC genes appear to be transcribed at the same time in the cell cycle, whereas in meiosis their transcripts accumulate with different kinetics, suggesting either that they have different turnover rates in meiotic cells or that the timing of their transcription is different.

Actins↗