Growth stage-based phenotypic analysis of Arabidopsis: a model for high throughput functional genomics in plants.

With the completion of the Arabidopsis genome sequencing project, the next major challenge is the large-scale determination of gene function. As a model organism for agricultural biotechnology, Arabidopsis presents the opportunity to provide key insights into the way that gene function can affect commercial crop production. In an attempt to aid in the rapid discovery of gene function, we have established a high throughput phenotypic analysis process based on a series of defined growth stages that serve both as developmental landmarks and as triggers for the collection of morphological data. The data collection process has been divided into two complementary platforms to ensure the capture of detailed data describing Arabidopsis growth and development over the entire life of the plant. The first platform characterizes early seedling growth on vertical plates for a period of 2 weeks. The second platform consists of an extensive set of measurements from plants grown on soil for a period of approximately 2 months. When combined with parallel processes for metabolic and gene expression profiling, these platforms constitute a core technology in the high throughput determination of gene function. We present here analyses of the development of wild-type Columbia (Col-0) plants and selected mutants to illustrate a framework methodology that can be used to identify and interpret phenotypic differences in plants resulting from genetic variation and/or environmental stress.

A specific, sensitive and high-capacity immunoassay for PAF.

A specific radioimmunoassay for platelet-activating factor (PAF) sensitive in the range 10-1000 pg (0.02-2 pmoles) has been developed. Detailed quantitative hapten inhibition studies showed specificity for the acetyl group at C-2 of PAF, a requirement for the ether linkage at C-1 and some tolerance for substituents on the choline nitrogen. No significant cross-reactivity was found with phosphatidylcholine and lysophosphatidylcholine or with lysoPAF.

A specific, sensitive radioimmunoassay for platelet-activating factor (PAF).

A specific radioimmunoassay (RIA) has been developed for platelet-activating factor (PAF) and shown to be sensitive over the range 10-1000 pg (0.02-2 pmol). The anti-PAF antibodies showed specificity for the acetyl group at the C2 position of the PAF molecule and exhibited no significant cross-reactivity with lyso-PAF or the naturally occurring lipids including lecithin and lysolecithin. The sensitivity of the RIA was at least as good as the platelet-based assays for PAF but the RIA was simpler to perform, had a higher capacity and did not have the drawback of the inherent variability associated with the bioassays.

Anaphylaxis following intranasal challenge of mice sensitized with ovalbumin.

Mice sensitized with two intraperitoneal injections of ovalbumin and challenged intranasally with the same antigen developed a non-fatal anaphylactic shock peaking in severity 30 min after challenge. Increases in haematocrit were noted which corresponded to the severity of signs of shock displayed by mice. Severity of shock also correlated with IgE and IgG levels. Sensitization by the nasal route, and use of B. pertussis vaccine as adjuvant had no qualitative effect upon the response. Cobra venom factor depletion of C3 in vivo did not alter the response of mice, which suggests anaphylaxis did not involve complement activation. Sensitivity was not transferrable to non-immune mice with serum. Passive sensitization with polyclonal and monoclonal antibodies produced inconsistent results. Possible mechanisms of anaphylaxis are discussed.

IgE and haemagglutinating antibody production in mice following intranasal immunization with ryegrass pollen.

Repeated intranasal immunization of mice whole ryegrass pollen (RGP) led to the development of high circulating titres of both IgE and haemagglutinating antibodies. Bordetella pertussis vaccine (BPV) was required at the time of initial immunization, although its repeated use did not affect IgE production. Antibody production was maintained at high levels for greater than 4 months. Significant differences were found in the ability of different batches and strains of BPV to induce production.

Enrichment and expansion of specific antibody-forming cells by adoptive transfer and clustering, and their use in hybridoma production.

Adoptive transfer regimens have been examined as a method of enriching and expanding antibody-forming cells (AFC). When spleens from mice which had reverted to memory or from those at the peak of an AFC response were transferred to syngeneic irradiated recipients, a comparable enrichment in AFC of about 10-fold was found. However, recently re-stimulated spleen cells gave much better expansion of total AFC in the recipient mice. The degree of expansion was examined using different routes and timing of antigen stimulus and AFC recovery. With the optimum protocol found the AFC pool obtained from adoptively-transferred recipients was on average 80-fold greater than from conventionally re-immunised mice in a number of experiments. Further enrichment of the AFC was shown by an in vitro clustering technique which gave suspensions with AFC enriched to better than 1 cell in 10. Cluster-enriched and adoptive-transfer enriched populations were both shown to give a much higher incidence of successful specific hybridoma production than spleen cells from conventionally re-immunised mice.