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PubMed2000

Technical advance: display and isolation of transposon-flanking sequences starting from genomic DNA or RNA.

Insertion mutagenesis using transposons has become a powerful tool for the isolation of genes involved in any given biochemical or developmental pathway. We describe here ligation-mediated PCR techniques for the isolation of sequences flanking the transposable elements En/Spm, Mu1 and Cin4 in Zea mays and Arabidopsis thaliana. Two versions of this transposon insertion display (TID) method use biotinylated linkers or biotinylated primers to rapidly isolate transposon-flanking sequences starting from digested genomic DNA. TID protocols have been employed to clone several genes from En/Spm insertion mutants of Arabidopsis. A novel procedure, expression TID (ETID), is also introduced, which provides a direct approach for the isolation of transposon insertions that tag transcribed portions of genes. ETID uses RNA as a starting material and exploits 5' RACE PCR to identify transposon copies that form parts of gene transcripts. The detection of several En/Spm insertion mutations in Arabidopsis illustrates the power of this method. ETID offers important advantages for the isolation of mutant alleles of novel genes that are expressed in specific tissues in plants and animals.

Alleles↗
PubMed2000

Technical advance: stringent control of transgene expression in Arabidopsis thaliana using the Top10 promoter system.

We show that the tightly regulated tetracycline-sensitive Top10 promoter system (Weinmann et al. Plant J. 1994, 5, 559-569) is functional in Arabidopsis thaliana. A pure breeding A. thaliana line (JL-tTA/8) was generated which expressed a chimeric fusion of the tetracycline repressor and the activation domain of Herpes simplex virus (tTA), from a single transgenic locus. Plants from this line were crossed with transgenics carrying the ER-targeted green fluorescent protein coding sequence (mGFP5) under control of the Top10 promoter sequence. Progeny from this cross displayed ER-targeted GFP fluorescence throughout the plant, indicating that the tTA-Top10 promoter interaction was functional in A. thaliana. GFP expression was repressed by 100 ng ml-1 tetracycline, an order of magnitude lower than the concentration used previously to repress expression in Nicotiana tabacum. Moreover, the level of GFP expression was controlled by varying the concentration of tetracycline in the medium, allowing a titred regulation of transgenic activity that was previously unavailable in A. thaliana. The kinetics of GFP activity were determined following de-repression of the Top10:mGFP5 transgene, with a visible ER-targeted GFP signal appearing from 24 to 48 h after de-repression.

Arabidopsis↗
PubMed2000

Technical advance: a high throughput system for transposon tagging and promoter trapping in tomato.

We describe new tools for functional analysis of the tomato genome based on insertional mutagenesis with the maize Ac/Ds transposable elements in the background of the miniature cultivar Micro-Tom. 2932 F3 families, in which Ds elements transposed and were stabilized, were screened for phenotypic mutations. Out of 10 families that had a clear mutant phenotype, only one mutant was Ds-tagged. In addition, we developed promoter trapping using the firefly luciferase reporter gene and enhancer trapping, using beta-glucuronidase (GUS). We show that luciferase can be used as a non-invasive reporter to identify, isolate and regenerate somatic sectors, to study the time course of mutant expression, and to identify inducible genes. Out of 108 families screened for luciferase activity 55% showed expression in the flower, 11% in the fruit and 4% in seedlings, suggesting a high rate of Ds insertion into genes. Preferential insertion into genes was supported by the analysis of Ds flanking sequences: 28 out of 50 sequenced Ds insertion sites were similar to known genes or to ESTs. In summary, the 2932 lines described here contain 2-3 Ds inserts per line, representing a collection of approximately 7500 Ds insertions. This collection has potential for use in high-throughput functional analysis of genes and promoter isolation in tomato.

DNA Transposable Elements↗
PubMed2000

Technical advance: simultaneous analysis of metabolites in potato tuber by gas chromatography-mass spectrometry.

A new method is presented in which gas chromatography coupled to mass spectrometry (GC-MS) allows the quantitative and qualitative detection of more than 150 compounds within a potato tuber, in a highly sensitive and specific manner. In contrast to other methods developed for metabolite analysis in plant systems, this method represents an unbiased and open approach that allows the detection of unexpected changes in metabolite levels. Although the method represents a compromise for a wide range of metabolites in terms of extraction, chemical modification and GC-MS analysis, for 25 metabolites analysed in detail the recoveries were found to be within the generally accepted range of 70-140%. Further, the reproducibility of the method was high: the error occurring in the analysis procedures was found to be less than 6% for 30 out of 33 compounds tested. Biological variability exceeded the systematic error of the analysis by a factor of up to 10. The method is also suited for upscaling, potentially allowing the simultaneous analysis of a large number of samples. As a first example this method has been applied to soil- and in vitro-grown tubers. Due to the simultaneous analysis of a wide range of metabolites it was immediately apparent that these systems differ significantly in their metabolism. Furthermore, the parallel insight into many pathways allows some conclusions to be drawn about the underlying physiological differences between both tuber systems. As a second example, transgenic lines modified in sucrose catabolism or starch synthesis were analysed. This example illustrates the power of an unbiased approach to detecting unexpected changes in transgenic lines.

Gas Chromatography-Mass Spectrometry↗
PubMed2000

Technical advance: the DNA-binding activity of gal4 is inhibited by methylation of the gal4 binding site in plant chromatin.

Derivatives of the Saccharomyces cerevisiae transcription factor Gal4 which act as effective transcription activators in yeast, Drosophila, mammalian cells and plant protoplasts are shown to direct expression from a GUS reporter construct when expressed in transgenic tobacco. However, in comparison to 35S-GUS controls, Gal4-mediated expression of the reporter gene was relatively weak and extremely variable. GUS expression was lost as plants matured and it was almost undetectable in most of their progeny. Gal4-mediated gene expression could be restored by treating tissues with 5-aza-cytidine, implicating cytosine methylation in the loss of Gal4-mediated expression. Restoration of reporter expression was not accompanied by an increase in steady-state levels of the activator transcript. We propose that the DNA-binding activity of Gal4 is sensitive to methylation of its binding site in plant chromatin. The Gal4-DNA co-crystal predicts that 5-methylcytosine at either of the outer two positions of the binding site will effectively prevent Gal4 binding. We show that these positions become extensively methylated in transgenic plants and that methylation of Gal4-binding sites interferes with Gal4 binding in vitro. These observations suggest that the Gal4 DNA-binding domain is intrinsically sensitive to cytosine methylation and that, despite the success of Gal4-based expression systems in yeast and Drosophila, Gal4 is not ideal for use in plant gene expression technology.

Azacitidine↗
PubMed2000

Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography.

Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

Actins↗
PubMed2000

Technical advance: An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants.

We have developed an estrogen receptor-based chemical-inducible system for use in transgenic plants. A chimeric transcription activator, XVE, was assembled by fusion of the DNA-binding domain of the bacterial repressor LexA (X), the acidic transactivating domain of VP16 (V) and the regulatory region of the human estrogen receptor (E; ER). The transactivating activity of the chimeric XVE factor, whose expression was controlled by the strong constitutive promoter G10-90, was strictly regulated by estrogens. In transgenic Arabidopsis and tobacco plants, estradiol-activated XVE can stimulate expression of a GFP reporter gene controlled by the target promoter, which consists of eight copies of the LexA operator fused upstream of the -46 35S minimal promoter. Upon induction by estradiol, GFP expression levels can be eightfold higher than that transcribed from a 35S promoter, whereas the uninduced controls have no detectable GFP transcripts, as monitored by Northern blot analysis. Neither toxic nor adverse physiological effects of the XVE system have been observed in transgenic Arabidopsis plants under all the conditions tested. The XVE system thus appears to be a reliable and efficient chemical-inducible system for regulating transgene expression in plants.

Arabidopsis↗
PubMed2000

Technical advance: an aniline blue staining procedure for confocal microscopy and 3D imaging of normal and perturbed cellular phenotypes in mature Arabidopsis embryos.

A new method is described for fluorescent imaging of mature Arabidopsis embryos that enables their cellular architecture to be visualized without the need for histological sectioning. Mature embryos are stained with aniline blue and cleared with chloral hydrate to allow high-resolution confocal imaging of individual cells within the embryo prior to germination. The technique allows the collection of longitudinal optical sections throughout the cotyledon, hypocotyl and root of wild-type Arabidopsis C24 embryos. Every cell within the mature embryo can be visualized with sufficient clarity and resolution to allow three-dimensional analysis of cellular architecture. Optical sectioning of mutant gnom, short-root and scarecrow embryos, and through root meristems disrupted as a consequence of targeted misexpression of diphtheria toxin, demonstrate the potential of this technique for visualizing the cellular organization of mutant and perturbed embryonic phenotypes.

Aniline Compounds↗
PubMed2000

Technical advance: application of high-performance liquid chromatography with photodiode array detection to the metabolic profiling of plant isoprenoids.

The application of high-performance liquid chromatography (HPLC) using a C30 reverse-phase stationary matrix has enabled the simultaneous separation of carotenes, xanthophylls, ubiquinones, tocopherols and plastoquinones in a single chromatogram. Continuous photodiode array (PDA) detection ensured identification and quantification of compounds upon elution. Applications of the method to the characterization of transgenic and mutant tomato varieties with altered isoprenoid content, biochemical screening of Arabidopsis thaliana, and elucidation of the modes of action of bleaching herbicides are described to illustrate the versatility of the procedure.

Arabidopsis↗
PubMed2000

Technical advance. Double-stranded RNA interferes with gene function at the single-cell level in cereals.

Double-stranded RNA (dsRNA) has been shown to specifically interfere with gene function in several organisms including tobacco and the model plant Arabidopsis. Here, we report on rapid and sequence-specific interference of dsRNA with gene function in cereals. Delivery of cognate dsRNA into single epidermal cells of maize, barley or wheat by particle bombardment interfered with the function of co-bombarded UidA (GUS) and TaGLP2a:GFP reporter genes. Cognate dsRNA was also found to specifically interfere with the function of the endogenous genes A1 and Ant18 encoding dihydroflavonol-4-reductase in maize and barley, respectively. Dihydroflavonol-4-reductase is an essential enzyme of the anthocyanin biosynthetic pathway in maize and barley. This pathway can be induced by transient expression of the C1- and b-Peru genes that encode transcription factors. In the presence of dsRNA corresponding to the dihydroflavonol-4-reductase gene, C1- and b-Peru-dependent, cell-autonomous accumulation of red anthocyanin pigments in bombarded cells of maize and barley was reduced. dsRNA was also demonstrated to negatively interfere with Mlo, which encodes a negative regulator of race non-specific resistance to the powdery mildew fungus in barley. In the presence of Mlo dsRNA, transformed cells became more resistant, thereby phenocopying plants that carry a heritable loss-of function mlo resistance allele. The results suggest that direct delivery of dsRNA to cereals leads to a rapid and sequence-specific interference with gene function at the single-cell level.

Alcohol Oxidoreductases↗
PubMed2001

Technical Advance: a novel technique for the sensitive quantification of acyl CoA esters from plant tissues.

We report a novel, highly sensitive and selective method for the extraction and quantification of acyl CoA esters from plant tissues. The method detects acyl CoA esters with acyl chain lengths from C4 to C20 down to concentrations as low as 6 fmol in extracts. Acyl CoA esters from standard solutions or plant extracts were derived to their fluorescent acyl etheno CoA esters in the presence of chloroacetaldehyde, separated by ion-paired reversed-phase high-performance liquid chromatography, and detected fluorometrically. This derivitization procedure circumvents the selectivity problems associated with previously published enzymatic methods, and methods that rely on acyl chain or thiol group modification for acyl CoA ester detection. The formation of acyl etheno CoA esters was verified by mass spectrometry, which was also used to identify unknown peaks from chromatograms of plant extracts. Using this method, we report the composition and concentration of the acyl CoA pool during lipid synthesis in maturing Brassica napus seeds and during storage lipid breakdown in 2-day-old Arabidopsis thaliana seedlings. The concentrations measured were in the 3--6 microM range for both tissue types. We also demonstrate the utility of acyl CoA profiling in a transgenic B. napus line that has high levels of lauric acid. To our knowledge, this is the first time that reliable estimates of acyl CoA ester concentrations have been made for higher plants, and the ability to profile these metabolites provides a valuable new tool for the investigation of gene function.

Acyl Coenzyme A↗
PubMed2002

Technical advances and pitfalls on the way to human cloning.

There exists a widespread consensus that the cloning of human beings to term would be detrimental to both the mother and child and of little value to society. However, the ambition of a few organisations and the recent advances in cellular and molecular technologies that led to the cloning of Dolly the sheep, for example, have meant that such a procedure will be possible if not illegal in the near future. The science associated with the cloning technologies practiced in other mammalian species reported to date provide important advances in our understanding of how cells function during early developmental processes and commit themselves to specific developmental pathways. However, many technological insufficiencies remain. Both technological advances and several of the associated insufficiencies are outlined in this review.

Animals↗
PubMed1988

Mechanical lithotripsy of bile duct stones in 209 patients--effect of technical advances.

Between 1982 and 1987, mechanical lithotripsy of common bile duct (CBD) stones was performed in 209 patients (mean age +/- SD: 72 +/- 10 years), using self-constructed lithotripters and baskets with breaking strengths of approximately 50 and 100 kg between 1982 and 1986, and stronger and bigger baskets with breaking strengths of approximately 125 kg since 1987. Patients with single (49.3%) and multiple stones (50.7%) were distributed about equally. Stone size ranged between 4 and 80 mm (mean longitudinal diameter 18 +/- 9 mm, mean transversal diameter 13 +/- 3 mm), with a majority of the patients (61.2%) having stones of between 10 and 19 mm, and about one-third (32.1%) of the patients with stones greater than or equal to 20 (20-80) mm. The overall success rate of mechanical lithotripsy in all 209 patients treated between 1982 and 1987 was 87.6% including 79.1% for CBD stones greater than or equal to 20 mm and 67.6% for stones greater than or equal to 25 mm. The introduction of baskets with breaking strengths of approximately 125 kg in 1987 considerably increased the success rate of mechanical lithotripsy, especially for "large and giant" CBD stones, to 92.3% (greater than or equal to 20 mm) and 85.7% (greater than or equal to 25 mm), respectively.

Aged↗
PubMed2003

New technical advances in swivel walkers.

Swivel walkers were commonly prescribed for children with complete thoracic lesion myelomeningocele in the 1970s and 80s, when the incidence of spina bifida in the UK was of the order of 3 per 100,000 live births. The advent of reciprocal walking orthoses provided a more suitable alternative for those with good upper limb and trunk function, and swivel walkers were then used primarily for very young or more severely disabled patients. Pre-natal screening has dramatically reduced the incidence of spina bifida in the UK and subsequently swivel walkers have been used in a wider range of pathology, including spinal muscular atrophy, multiple sclerosis, muscular dystrophy and other neurological conditions that lead to lower limb dysfunction. The detail design of these devices has been adapted to accommodate the specific problems encountered in these conditions. In particular the designs have been updated to: enable very young patients to be more readily fitted at the age of 1 year; allow the walking mechanisms to be conveniently adjusted for easier ambulation when weakness or lack of confidence inhibits performance; permit simple adjustment to a standing frame mode to enhance stability in situations of increased risk; promote manual handling practice that is compatible with the National Health Service (NHS) policy of compliance with relevant regulations. To underpin appropriate prescription and safe supply the NHS Procurement Agency have encouraged the development of a common course for all types of swivel walker.

Adolescent↗
PubMed2026

The Gyrolab platform for immunogenicity assessment and biotherapeutic and biomarker analysis: technical advances and bioanalytical applications.

Gyrolab is an automated ligand‑binding assay platform designed for the quantification of proteins with high sensitivity and broad dynamic range. Its low minimum required dilution and microfluidic disc format enable efficient sample processing while maintaining assay robustness, making it a valuable tool across drug‑development stages. This review summarizes published applications of Gyrolab for pharmacokinetic and toxicokinetic analysis and immunogenicity assessment through anti‑drug antibody detection. In addition, the platform's high-volume assay discs have facilitated its use in biomarker studies, allowing quantification of low‑abundance analytes. Representative examples from the literature are presented together with key assay elements, including analytes, matrices, dynamic ranges, and critical reagents. Because assay replicates remain an important consideration for Gyrolab workflows, recent publications addressing singlet versus duplicate strategies and their impact on data interpretation are also discussed. Finally, we provide real analytical data visualized using a three‑dimensional Gyrolab viewer to illustrate variability in duplicate measurements and to highlight future opportunities for improving assay reliability.

Humans↗
PubMed2003

Technical advances: genome-wide cDNA-AFLP analysis of the Arabidopsis transcriptome.

cDNA-AFLP, a technology historically used to identify small numbers of differentially expressed genes, was adapted as a genome-wide transcript profiling method. mRNA levels were assayed in a diverse range of tissues from Arabidopsis thaliana plants grown under a variety of environmental conditions. The resulting cDNA-AFLP fragments were sequenced. By linking cDNA-AFLP fragments to their corresponding mRNAs via these sequences, a database was generated that contained quantitative expression information for up to two-thirds of gene loci in A. thaliana, ecotype Ws. Using this resource, the expression levels of genes, including those with high nucleotide sequence similarity, could be determined in a high-throughput manner merely by comparing cDNA-AFLP profiles with the database. The lengths of cDNA-AFLP fragments inferred from their electrophoretic mobilities correlated well with actual fragment lengths determined by sequencing. In addition, the concentrations of AFLP fragments from single cDNAs were highly correlated, illustrating the validity of cDNA-AFLP as a quantitative, genome-wide, transcript profiling method. cDNA-AFLP profiles were also qualitatively consistent with mRNA profiles obtained from parallel microarray analysis, and with data from previous studies.

Arabidopsis↗