Using degenerate oligonucleotide primers based on a pumpkin (Cucurbita maxima) gibberellin (CA) 20-oxidase sequence, six different fragments of dioxygenase genes were amplified by polymerase chain reaction from Arabidopsis fhaliana genomic DNA. One of these was used to isolate two different full-length cDNA clones, At2301 and At2353, from shoots of the CA-deficient Arabidopsis mutant gal-2. A third, related clone, YAP169, was identified in the Database of Expressed Sequence Tags. The cDNA clones were expressed in Escherichia coli as fusion proteins, each of which oxidized CA,, at C-20 to GA,,, CAZ4, and the C,, compound CA,, a precursor of bioactive CAs; the C, , tricarboxylic acid compound CA,, was formed as a minor product. The expression products also oxidized the 13-hydroxylated substrate CA,,, but less effectively than CA,,. The three cDNAs hybridized to mRNA species with tissue-specific patterns of accumulation, with At2301 being expressed in stems and inflorescences, At2353 in inflorescences and developing diques, and YAPl69 in diques only. In the floral shoots of the gal-2 mutant, transcript levels corresponding to each cDNA decreased dramatically after CA, application, suggesting that CA biosynthesis may be controlled, at least in part, through downregulation of the expression of the 20-oxidase genes.

Objective: Current cerebrospinal fluid (CSF) tests for sporadic Creutzfeldt–Jakob disease (sCJD) are based on the detection of surrogate markers of neuronal damage such as CSF 14‐3‐3, which are not specific for sCJD. A number of prion protein conversion assays have been developed, including real time quaking‐induced conversion (RT‐QuIC). The objective of this study is to investigate whether CSF RT‐QuIC analysis could be used as a diagnostic test in sCJD. Methods: An exploratory study was undertaken that analyzed 108 CSF samples from patients with neuropathologically confirmed sCJD or from control patients. Of the 108 CSF samples, 56 were from sCJD patients (30 female, 26 male; aged 31–84 years; mean age, 62.3 ± 13.5 years), and 52 were from control patients (26 female, 26 male; aged 43–84 years; mean age, 67.8 ± 10.4 years). A confirmatory group of 118 patients was subsequently examined that consisted of 67 cases of neuropathologically confirmed sCJD (33 female, 34 male; aged 39–82 years; mean age, 67.5 ± 9.0 years) and 51 control cases (26 female, 25 male; aged 36–87 years; mean age, 63.5 ± 11.6 years). Results: The exploratory study showed that RT‐QuIC analysis had a sensitivity of 91% and a specificity of 98% for the diagnosis of sCJD. These results were confirmed in the confirmatory study, which showed that CSF RT‐QuIC analysis had a sensitivity and specificity of 87% and 100%, respectively. Interpretation: This study shows that CSF RT‐QuIC analysis has the potential to be a more specific diagnostic test for sCJD than current CSF tests. ANN NEUROL 2012;72:278–285.

A monoclonal antibody produced to abscisic acid (ABA) has been characterised and the development of a radioimmunoassay (RIA) for ABA using the antibody is described. The antibody had a high selectivity for the free acid of (S)-cis, trans-ABA. Using the antibody, ABA could be assayed reliably in the RIA over a range from 100 to 4000 pg (0.4 to 15 pmol) ABA per assay vial. As methanol and acetone affected ABA-antibody binding, water was used to extract ABA from leaves. Water was as effective as aqueous methanol and acetone in extracting the ABA present. Crude aqueous extracts of wheat, maize and lupin leaves could be analysed without serious interference from other immunoreactive material. This was shown by measuring the distribution of immunoreactivity in crude extracts separated by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), or by comparing the assay with physicochemical methods of analysis. Analysis of crude extracts by RIA and either, after TLC purification, by gas chromatography using an electron-capture detector or, after HPLC purification, by combined gas chromatography-mass spectrometry (GC-MS) gave very similar ABA concentrations in the initial leaf samples. However, RIA analysis of crude aqueous extracts of pea seeds resulted in considerable overestimation of the amount of ABA present. Determinations of ABA content by GC-MS and RIA were similar after pea seed extracts had been purified by HPLC. Although the RIA could not be used to analyse ABA in crude extracts of pea seeds, it is likely that crude extracts of leaves of several other species may be assayed successfully.

Real-time quaking-induced conversion (RT-QuIC) is an assay in which disease-associated prion protein (PrP) initiates a rapid conformational transition in recombinant PrP (recPrP), resulting in the formation of amyloid that can be monitored in real time using the dye thioflavin T. It therefore has potential advantages over analogous cell-free PrP conversion assays such as protein misfolding cyclic amplification (PMCA). The QuIC assay and the related amyloid seeding assay have been developed largely using rodent-passaged sheep scrapie strains. Given the potential RT-QuIC has for Creutzfeldt-Jakob disease (CJD) research and human prion test development, this study characterized the behaviour of a range of CJD brain specimens with hamster and human recPrP in the RT-QuIC assay. The results showed that RT-QuIC is a rapid, sensitive and specific test for the form of abnormal PrP found in the most commonly occurring forms of sporadic CJD. The assay appeared to be largely independent of species-related sequence differences between human and hamster recPrP and of the methionine/valine polymorphism at codon 129 of the human PrP gene. However, with the same conditions and substrate, the assay was less efficient in detecting the abnormal PrP that characterizes variant CJD brain. Comparison of these QuIC results with those previously obtained using PMCA suggested that these two seemingly similar assays differ in important respects.

The enzymes gibberellin (GA) 20-oxidase and 3-oxidase are major sites of regulation in GA biosynthesis. We have characterised one member of each of the gene families encoding these enzymes that are highly expressed in elongating stems and in developing and germinating grains of wheat and are therefore likely to have prominent developmental roles in these tissues. We mapped the three homoeologues of the GA 20-oxidase gene TaGA20ox1 to chromosomes 5BL, 5DL and 4AL. TaGA20ox1 is expressed mainly in the nodes and ears of the elongating stem, and also in developing and germinating embryos. Expression in the nodes, ears and germinating embryos is predominantly from the A and D genomes. Each homoeologous cDNA encodes a functional enzyme that catalyses the multi-step conversions of GA12-GA9, and GA53-GA20. Time course and enzyme kinetic studies indicate that the initial oxidation steps from GA12 and GA53 to the free alcohol forms of GA15 and GA44, respectively, occur rapidly but that subsequent steps occur more slowly. The intermediate GA19 has an especially low affinity for the enzyme, consistent with its accumulation in wheat tissues. The three homoeologous cDNAs for the 3-oxidase gene TaGA3ox2 encode functional enzymes, one of which was shown to possess low levels of 2beta-hydroxylase, 2,3-desaturase, 2,3-epoxidase and even 13-hydroxylase activities in addition to 3beta-hydroxylase activity. In contrast to TaGA20ox1, TaGA3ox2 is expressed in internodes, as well as nodes and the ear of the elongating stem. It is also highly expressed in developing and germinated embryos.

Ectopic expression of a gibberellin 2-oxidase gene (PcGA2ox1) decreased the content of bioactive gibberellins (GAs) in transgenic wheat, producing a range of dwarf plants with different degrees of severity. In at least one case, a single transformation event gave rise to T(1) plants with different degrees of dwarfism, the phenotypes being stably inherited over at least four generations. The dwarf phenotype, which included dark-green leaves, increased tillering and, in severe cases, a prostrate growth habit, was replicated by the application of a GA biosynthesis inhibitor to the wild type. Ear rachis length, grain set, and grain size were also decreased in the wheat transformants, compared with an azygous (null) line. The extent of post-germination alpha-amylase production in grains reflected the severity of the shoot phenotype of the transformants and both developmental processes were restored to normal by the application of gibberellic acid (GA(3)). Expression of two GA biosynthesis genes (TaGA20ox1 and TaGA3ox2) was up-regulated, and that of two alpha-amylase gene families (alpha-Amy1 and alpha-Amy2) down regulated, in scutella of semi-dwarf lines, compared with controls. The marked decline in transcript abundance of both alpha-amylase gene families in aleurone was associated with a decreased content of bioactive GAs in grains of the semi-dwarf lines.

The diffusion of GA1and GA3from the embryo, and the decline in ABA content of endosperm, were associated with the induction of α‐amylase (EC 3.2.1.1) gene expression in aleurone of intact wheat (Triticum aestivum L. cv. Maris Huntsman) grains germinated at 25°C. The scutellum appeared to be the main site of de novo GA biosynthesis based on (1) the abundance of transcripts of a cloned wheat GA 20‐oxidase. (2) the increase in content of GAs belonging to the early 13‐hydroxylation GA pathway, and (3) the accumulation of ent‐kaurene in grains imbibed in the presence of an ent‐kaurene oxidase inhibitor. Again, the initiation of GA biosynthesis in the scutellum was closely associated with the induction of α‐amylase gene expression in scutellar epithelium, although the two events may not have been causally linked. The embryo was required to be present for 36 h from the start of imbibition in order to induce α‐amylase activity in aleurone, and the response could be replicated by low doses of GA1applied to de‐embryonated grains. After‐ripened wheat aleurone was relatively unresponsive to applied ABA in terms of suppression of GA‐induced α‐amylase production. Subtle differences were observed in the temporal pattern of α‐amylase gene expression between intact germinated grains and de‐embryonated grains challenged with GA1. It appears that endogenous GAs are an important component of the embryo stimulus initiating α‐amylase gene expression in aleurone of germinating wheat grain, as originally proposed for barley. Their role in the synthesis of α‐amylase in scutellar epithelium remains to be clarified.

In near-isogenic lines of winter wheat (Triticum aestivum L. cv. Maris Huntsman) grown at 20° C under long days the reduced-height genes, Rht1 (semi-dwarf) and Rht3 (dwarf) reduced the rate of extension of leaf 2 by 12% and 52%, respectively, compared with corresponding rht (tall) lines. Lowering the growing temperature from 20° to 10° C reduced the rate of linear extension of leaf 2 by 2.5-fold (60% reduction) in the rht3 line but by only 1.6-fold (36% reduction) in the Rht3 line. For both genotypes, the duration of leaf expansion was greater at the lower temperature so that final leaf length was reduced by only 35% in the rht3 line and was similar in the Rht3 line at both temperatures. Seedlings of the rht3 (tall) line growing at 20° C responded positively to root-applied gibberellin A1 (GA1) in the range 1-10 μM GA1; there was a linear increase in sheath length of leaf 1 whereas the Rht3 (dwarf) line remained unresponsive. Gibberellins A1, 3, 4, 8, 19, 20, 29, 34, 44 and 53 were identified by full-scan gas chromatography-mass spectrometry in aseptically grown 4-d-old shoots of the Rht3 line. In 12-d-old seedlings grown at 20° C, there were fourfold and 24-fold increases in the concentration of GA1 in the leaf expansion zone of Rht1 and Rht3 lines, respectively, compared with corresponding rht lines. Although GA3 was present at a similar level to GA1 in the rht3 (tall) line it accumulated only fivefold in the Rht3 (dwarf) line. The steady-state pool sizes of endogenous GAs were GA19 ≫ GA20 = GA1 in the GA-responsive rht3 line whereas in the GA non-responsive Rht3 line the content of GA19≈ GA20 ⋘ GA1. It is proposed that one of the consequences of GA1 action is suppression of GA19-oxidase activity such that the conversion of GA19 to GA20 becomes a rate-limiting step on the pathway to GA1 in GA-responsive lines. In the GA-non-responsive Rht lines it is suggested that GA19 oxidase is not downregulated to the same extent and GA1 accumulates before the next rate-limiting step on the pathway, its 2β-hydroxylation to GA8. The steady-state pool sizes of GA19, 20, 1, 3 and 8 were similar in developmentally equivalent tissues of the rht3 (tall) line growing at 10° C and 20° C despite a 2.5-fold difference in the rate of leaf expansion. In contrast, in the Rht3 (dwarf) line, the extent of accumulation of GA1 reflected the severity of the phenotype at the two temperatures with slower growing tissues accumulating less, not more, GA1. These results are interpreted as supporting the proposed model of regulation of the GA-biosynthetic pathway rather than previous suggestions that GA1 accumulates in GA-insensitive dwarfs as a consequence of reduced growth rates.