FMD control strategies.
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Animal Welfare↗
Biomedical subjects
T H Ellis
Publications and source records attributed to T H Ellis.
Stability and inheritance of methylation states at PstI sites in Pisum.
The nuclear genome of pea is heavily methylated and the stability of this methylation pattern is unknown. In this study we investigated the stability of DNA methylation and its contribution to restriction fragment polymorphism as judged by AFLP fragment differences. To do this the results of a conventional AFLP analysis were compared with those obtained by a related procedure performed on pre-amplified DNA which provided a 5-methylcytosine-free DNA template (SDAFLP). Genetic mapping in a recombinant inbred (RI) population showed that polymorphisms attributable to different methylation states of PstI sites were abundant, and generally appeared to be stably inherited, although occasional failures of the inheritance of methylation states have been found. Assessments of genetic diversity by AFLP and SDAFLP were in general agreement with each other and with the currently accepted phylogeny of Pisum, but within cultivated groups the number of differences appeared to be exaggerated by AFLP. The data suggest that epigenetic differences may have played a role in the domestication of pea.
5-Methylcytosine↗
A tapping mode AFM study of collapse and denaturation in dentinal collagen.
OBJECTIVES: Tapping mode atomic force microscopy (AFM) was used to investigate the surface changes of collagen exposed to phosphoric acid treatment. We focus on denaturation and collapse following demineralization and exposure to air. METHODS: Unpolished dentin disks, obtained from freshly extracted human molars, were etched in 37% phosphoric acid for 15s, rinsed ultrasonically and gently blotted with soft paper; the specimens were then continuously observed using tapping mode AFM. RESULTS: Immediately after the removal of bulk water, the surface consisted of a porous network of banded collagen fibrils, having periodicities of 67nm. After approximately 8min of subsequent air-drying, the spacing between fibrils was lost, and the surface was observed to consist of a dense array of closely spaced fibrils. The banding periodicity was still observable. SIGNIFICANCE: The air drying of etched dentin results in the collapse of the collagen network, but not in the denaturation of the collagen fibrils. This study indicates that collapse and denaturation are separate phenomena. It further shows that water loss occurs rapidly, and disrupts the native conformation of the collagen network. This would have adverse effects on adhesion.
Acid Etching, Dental↗
A photoacoustic FTIRS study of the chemical modifications of human dentin surfaces: II. Deproteination.
This work focuses on the chemical alterations occurring on the dentin surface on treatment with sodium hypochlorite (NaOCl), a known deproteinating agent. In addition, sequential treatment with both acid and NaOCl are characterized. Modifications are evaluated using photoacoustic FTIR (PA-FTIRS), a surface-sensitive spectroscopy. Spectra of NaOCl-treated dentin samples show a slow and heterogeneous removal of its organic phase, leaving calcium hydroxyapatite and carbonate apatite unchanged. Spectra of this deproteinated surface resemble those of apatites synthesized at low temperatures and having very high-surface areas. A combined sequential 2-min treatment of dentin with both maleic acid and NaOCl indicates that this treatment can produce a surface region which is neither significantly demineralized nor deproteinated. This sequential treatment can be used to remove the smear layer and restore the dentin surface to its natural composition.
Acid Etching, Dental↗
A photoacoustic FTIRS study of the chemical modifications of human dentin surfaces: I. Demineralization.
Acids are used to modify the structure and composition of dentin surfaces to improve bonds formed with resins. The purpose of this work is to investigate such chemical modifications using the surface-sensitive technique photoacoustic Fourier transform infrared spectroscopy (PA-FTIRS). Spectra of acid-treated samples (citric, maleic, nitric, and phosphoric at pH = 1.0) were recorded at various time intervals. Analysis of these spectra indicates a gradual increase in sample surface area with treatment time. A decrease of the bands associated with calcium hydroxyapatite (HAP) and carbonate apatite inherent to the mineral phase of dentin are also observed. A comparison of spectra of samples treated for 2 min with each acid also reveals that maleic and phosphoric acids remove more HAP than citric acid. We conclude that citric acid may cause the formation of precipitates at the etching front which inhibit etching.
Acids↗
Expression of a class 1 knotted1-like homeobox gene is down-regulated in pea compound leaf primordia.
Differences in knotted1-like (knox) gene expression may account for some of the diversity of leaf forms seen in nature. Class 1 knox genes are expressed in the compound leaf primordia of tomato but not in the simple leaf primordia of a range of species examined so far. In order to test the hypothesis that all compound leaves differ from simple leaves in this way, we isolated a class 1 knox cDNA from pea, Pskn1 (Pisum sativum knotted1) and examined its expression pattern. The encoded homeodomain of Pskn1 shares 88% identical residues with KNOTTED1 from maize and an adjacent ELK domain is present. The protein sequence of PSKN1 is 69% identical to TKN2, its nearest related sequence in tomato. Unlike TKn2, Pskn1 was not expressed in newly initiated compound leaves. The expression pattern of Pskn1 resembled those of other class 1 knox genes described in maize and Arabidopsis. Transcripts were detected in the shoot apical meristem and developing vasculature of the vegetative shoot, but expression was not detected in newly initiated and developing compound leaf primordia. The same pattern of expression was observed in the afila mutant, which is characterised by highly ramified compound leaves. Our results suggest that tomato and pea use different developmental processes in the generation of their compound leaves.
Amino Acid Sequence↗
Pea Ty1-copia group retrotransposons: transpositional activity and use as markers to study genetic diversity in Pisum.
The variation in transposition history of different Ty1-copia group LTR retrotransposons in the species lineages of the Pisum genus has been investigated. A heterogeneous population of Ty1-copia elements was isolated by degenerate PCR and two of these (Tps12 and Tps19) were selected on the basis of their copy number and sequence conservation between closely related species for further in-depth study of their transpositional history in Pisum species. The insertional polymorphism of these elements and the previously characterised PDR1 element was studied by sequence-specific amplification polymorphism (SSAP). Each of these elements reveals a unique transpositional history within 55 diverse Pisum accessions. Phylogenetic trees based on the SSAP data show that SSAP markers for individual elements are able to resolve different species lineages within the Pisum genus. Finally, the SSAP data from all of these retrotransposon markers were combined to reveal a detailed picture of the intra and interspecies relationships within Pisum.
Amino Acid Sequence↗
Nodule-expressed Cyp15a cysteine protease genes map to syntenic genome regions in Pisum and Medicago spp.
PsCyp15a is a gene that encodes a vacuolar cysteine protease expressed in wilt-induced shoots of Pisum sativum (pea) and in root nodules. To further the understanding of nodular PsCyp15a expression, a region 5' to the coding sequence of the gene was cloned. Varying lengths of 5' untranslated sequence were fused with the uidA coding region and introduced from Agrobacterium rhizogenes into "hairy roots" of Vicia hirsuta. In this transgenic root nodulation assay, a promoter sequence of 900 bp was sufficient to give an expression pattern indistinguishable from that obtained in pea nodules by in situ hybridization. An orthologue of PsCyp15a was cloned from nodule mRNA of Medicago sativa and a corresponding gene identified in M. truncatula was also shown to express strongly in nodules. With molecular mapping techniques, it was demonstrated that these genes map to a syntenic genome location in pea and Medicago spp., but the map positions of the Cyp15a genes cannot be correlated with existing nodulation mutants.
Amino Acid Sequence↗
Pea compound leaf architecture is regulated by interactions among the genes UNIFOLIATA, cochleata, afila, and tendril-lessn.
The compound leaf primordium of pea represents a marginal blastozone that initiates organ primordia, in an acropetal manner, from its growing distal region. The UNIFOLIATA (UNI) gene is important in marginal blastozone maintenance because loss or reduction of its function results in uni mutant leaves of reduced complexity. In this study, we show that UNI is expressed in the leaf blastozone over the period in which organ primordia are initiated and is downregulated at the time of leaf primordium determination. Prolonged UNI expression was associated with increased blastozone activity in the complex leaves of afila (af), cochleata (coch), and afila tendril-less (af tl) mutant plants. Our analysis suggests that UNI expression is negatively regulated by COCH in stipule primordia, by AF in proximal leaflet primordia, and by AF and TL in distal and terminal tendril primordia. We propose that the control of UNI expression by AF, TL, and COCH is important in the regulation of blastozone activity and pattern formation in the compound leaf primordium of the pea.
Epistasis, Genetic↗
Field emission in-lens SEM study of enamel and dentin.
This investigation used field emission in-lens scanning electron microscopy (FEISEM) for the study of tooth surfaces, with particular reference to adhesive bonding and acid conditioning. Dentin wafers with an intact enamel periphery were treated by either ethylenediaminetetraacetic acid (EDTA) (pH 7.4) or phosphoric acid (pH 0.7). The samples were then fixed, sequentially dehydrated in alcohol, and either air- or critical point-dried. After coating, surfaces were examined by FEISEM. For enamel, intraprismatic crystals were clearly recognizable, with the crystals showing both a longitudinal and parallel orientation to the long axis of the prisms. For dentin, the surface ultrastructure (mineral crystals and collagen banding) for the both untreated and treated samples was observed. Fine structures measuring on the order of 6 nm were also observed on samples treated by EDTA. We conclude that FEISEM can routinely provide high-resolution images of enamel and dentin, and that it has the capability of revealing the defined distribution of crystals and collagen fibers in dental tissues.
Acid Etching, Dental↗
Genetic mapping and functional analysis of a nodulation-defective mutant (sym19) of pea (Pisum sativum L.).
The pea mutant line P55 is defective in root nodule formation, and this phenotype is controlled by a single recessive gene. Complementation analysis revealed that the mutation in P55 is allelic to sym19, which has previously been mapped to linkage group I. Detailed mapping revealed that the sym19 and ENOD40 loci are separated by 2.7 cM. We identified four recombination events, demonstrating that the nodulation defect caused by mutation of the sym19 locus cannot be due to mutation of ENOD40. RT-PCR experiments showed that P55 expresses ENOD12A, but there was little or no increase in the level of its transcript in response to Nod factor or infection with Rhizobium. To investigate this expression pattern further, transgenic peas carrying a pENOD12A-GUS reporter construct were made. One transgenic line was crossed with line P55, to generate F2 progeny homozygous for sym19 and carrying pENOD12A-GUS. In both WT and sym19 mutant lines, ENOD12A-GUS expression was induced at sites of lateral root emergence in uninoculated plants. In Nod+ plants pENOD12A-GUS was induced in response to Rhizobium leguminosarumn bv. viciae, but no such induction was seen in the Nod- (sym19) mutants.
Alleles↗
Heterogeneity of the internal structure of PDR1, a family of Ty1/copia-like retrotransposons in pea.
We characterised the extent of heterogeneity among PDR1 elements, a Ty1/copia-like retrotransposon family in pea, by restriction mapping and PCR with primers designed to amplify four functional domains. The data suggest that two main subfamilies of PDR1 differ in the size of their 5'-region. There are also sequence variants and rearranged copies which include a wide range of deletions of different sizes and deletions combined with insertions of host DNA, or inversions of various regions of the retrotransposon. A deletion hotspot has been found at nucleotide position 394, where buffer sequences of 26 bp and 38 bp containing microsatellite motifs have been generated. There is more heterogeneity in the gag domain of PDR1 than in other functional domains, and the extent and pattern of this diversity was assessed among 56 Pisum accessions. We found a higher rate of rearrangement and sequence variation within the gag domain of PDR1 in P. fulvum and P. abyssinicum accessions than would be expected from the degree of insertion site polymorphism. A neighbour-joining phylogenetic tree constructed for gag sequences has a similar branching pattern to the equivalent insertion site tree, implying that the PDR1 family and its gag domain have coevolved with the pea genome. Combining both trees revealed clear and distinct subgroups among the Pisum ssp.
Base Sequence↗
Rapid isolation of plant Ty1-copia group retrotransposon LTR sequences for molecular marker studies.
The terminal sequences of long-terminal repeat (LTR) retrotransposons are a source of powerful molecular markers for linkage mapping and biodiversity studies. The major factor limiting the widespread application of LTR retrotransposon-based molecular markers is the availability of new retrotransposon terminal sequences. We describe a PCR-based method for the rapid isolation of LTR sequences of Ty1-copia group retrotransposons from the genomic DNA of potentially any higher plant species. To demonstrate the utility of this technique, we have identified a variety of new retrotransposon LTR sequences from pea, broad bean and Norway spruce. Primers specific for three pea LTRs have been used to reveal polymorphisms associated with the corresponding retrotransposons within the Pisum genus.
Amino Acid Sequence↗
Moisture-dependent renaturation of collagen in phosphoric acid etched human dentin.
We used atomic force microscopy (AFM) to investigate the effects of acidic and aqueous treatments on human dentin. Two basic points were determined: the first is the ability of AFM to discriminate the effect of phosphoric acid (pH approximately equal to 1) on polished dentin, and the second is the demonstrable effect of moisture on fibrous collagen structure. AFM images confirmed that the polishing process led to the removal of both smear layer and smear plugs. Our AFM study of undried dentin, which was then acid treated and kept moist, revealed substantial morphological changes at the dentin surface. Collagen fibers, having a characteristic periodicity of 67 nm, were imaged in situ for the first time; these structures were absent in dentin treated by phosphoric acid and subsequently vacuum dried, even after prolonged reimmersion in water. The AFM technique permitted us to demonstrate the important roles that moisture and etching play in the determination of the structure of collagen fibrils. Such structure may also play an important role in the diffusibility of subsequently applied dental adhesion systems.
Collagen↗
Polymorphism of insertion sites of Ty1-copia class retrotransposons and its use for linkage and diversity analysis in pea.
A sample of 15 cultivars and 56 Pisum accessions from the JIC germplasm core collection has been studied using a modification of the SSAP (sequence-specific amplification polymorphisms) technique; the specific primer was designed to correspond to the polypurine tract (PPT) of PDR1, a Ty1-copia group retrotransposon of pea. Most of these SSAP products were shown to be PDR1 derived. The PDR1 SSAP markers are more informative than previously studied AFLP or RFLP markers and are distributed throughout the genome. Their pattern of variation makes them ideal for integrating genetic maps derived from related crosses. Data sets obtained with AFLP and PDR1 SSAP markers were used to construct neighbour-joining trees and for principal component analysis. These data sets give greater resolution than hitherto available for the characterisation of variation within Pisum, showing that the genus has three main groups: P. fulvum, P. abyssinicum and all other Pisum spp. P. abyssinicum is not a subgroup of cultivated P. sativum, as was previously thought, but has probably been domesticated independently. Modern cultivars are shown to form a single group within Pisum as a whole.
Biological Evolution↗
Retrotransposon-based insertion polymorphisms (RBIP) for high throughput marker analysis.
Two assays based upon PCR detection of a polymorphic PDR1 retrotransposon insertion in Pisum sativum have been developed. Both methods involve PCR with primers derived from the transposon and flanking DNA. The first method uses a dot assay for PCR product detection which could be fully automated for handling thousands of samples. The second method, which is designed to handle lower numbers, requires a single PCR and gel lane per sample. Both methods yield co-dominant markers, with presence and absence of the transposon insertion independently scorable, and both could in principle be applied to any transposable element in any plant species.
Alleles↗
The pea early nodulin gene PsENOD7 maps in the region of linkage group I containing sym2 and leghaemoglobin.
The early nodulin gene, PsENOD7, is expressed in pea root nodules induced by Rhizobium leguminosarum bv. viciae, but not in other plant organs. In situ hybridization showed that this gene is transcribed during nodule maturation in the infected cells of the proximal part of the prefixation zone II. At the transition of zone II into interzone II-III, the level of PsENOD7 mRNA drops markedly. PsENOD7 has no significant homology to other genes. RFLP mapping studies have shown that PsENOD7 is located in linkage group I between the leghaemoglobin genes and sym2.
Amino Acid Sequence↗