R. Bruce Knox

Genetic control of self-incompatibility and reproductive development in flowering plants

A: Self-Incompatibility. I. Molecular biology. II. Cellular and whole plant studies. B: Control of Sex Expression and Floral Development. C: Genes Active in Male Gametogenesis: Pollen Genes. I. Cellular and molecular biology. II. Male sterility. D: Female Gametogenesis and Fertilization. Index.

Cloning sequencing of Lol pI, the major allergenic protein of rye-grass pollen

We have isolated a full length cDNA clone encoding the major glycoprotein allergen Lol pI. The clone was selected using a combination of immunological screening of a cDNA expression library and PCR amplification of Lol pI‐specific transcripts. Lol pI expressed in bacteria as a fusion protein shows recognition by specific IgE antibodies present in sera of grass pollen‐allergic subjects. Northern analysis has shown that the Lol pI transcripts are expressed only in pollen of rye‐grass. Molecular cloning of Lol pI provides a molecular genetic approach to study the structure—function relationship of allergens.

Concentrations of the major birch tree allergen Bet v 1 in pollen and respirable fine particles in the atmosphere

BACKGROUND Birch tree pollen allergens are an important cause of early spring hay fever and allergic asthma. Pollen counts provide a guide for individuals with birch pollen allergy. However, birch pollen, because of its size, has a low probability of entering the lower airways to trigger asthma. Yet birch pollen allergens are known to be associated with respirable particles present in the atmosphere. OBJECTIVE We sought to determine the concentration of major allergen Bet v 1 in birch pollen and respirable particles in the atmosphere during the birch pollen season. METHODS We used a two-site monoclonal antibody-based assay (ELISA) to quantitate Bet v 1 in pollen extracts and high-volume air sampler filters collecting particles larger and smaller than 7.2 microm. RESULTS Bet v 1 (0.006 ng) is detectable per birch pollen grain, of which 0.004 ng is present in aqueous extracts (13.9% of soluble proteins). Atmospheric Bet v 1 concentrations are correlated with birch pollen counts. Heavy rainfall tended to wash out pollen and particles, indicated by a mean daily Bet v 1 concentration of 0.12 ng/m3 (20 pollen equivalents), but light rainfall produced a dramatic increase in allergen-loaded respirable particles with Bet v 1 concentrations of 1.2 ng/m3 (200 pollen equivalents). CONCLUSION These results highlight the different environmental risk factors for hay fever and allergic asthma in patients sensitized to Bet v 1. Light rainfall causes an increase in respirable particles; hence, this is an important risk factor for asthma.

Molecular cloning and immunological characterisation of Cyn d 7, a novel calcium-binding allergen from Bermuda grass pollen.

A cDNA coding for a newly identified Bermuda grass pollen allergen, Cyn d 7, with significant sequence similarity to Ca2+‐binding proteins, was isolated from a cDNA expression library using serum IgE from an allergic individual. The deduced amino acid sequence of Cyn d 7 contained two typical Ca2+‐binding sites (EF hand domains). Depletion of Ca2+ with EGTA led to a loss of IgE‐binding capacity of rCyn d 7. A synthetic peptide based on domain II showed high IgE reactivity. Cyn d 7 therefore represents a grass pollen allergen that belongs to a novel class of Ca2+‐binding proteins.

Cell Recognition in Flowering Plants

Plant cells in general have a capacity to discriminate between self and non-self. The case for cell recognition in plants is made primarily on evidence from pollen-stigma interactions and stem-grafting experiments. Recognition results in a defined response and may be mediated by surface or secreted determinants, likely to be protein or glycoprotein in nature. Mediators may include lectins, cell wall components such as arabinogalactan proteins, arabinoxylans, and allergens. Antigenic cell determinants may provide an alternative to the traditional morphological approach for the typing of plant cells.

Cloning and expression in yeast Pichia pastoris of a biologically active form of Cyn d 1, the major allergen of Bermuda grass pollen

BACKGROUND Pollen of grasses, such as Bermuda grass (Cynodon dactylon), represent a major cause of type I allergy. OBJECTIVE In this report we attempted to clone and express a biologically active form of recombinant Cyn d 1, the major allergen of Bermuda grass pollen, in the yeast Pichia pastoris. METHODS Clones encoding Cyn d 1 were isolated by screening a Bermuda grass pollen complementary DNA library with specific monoclonal antibodies and by polymerase chain reaction amplification. Recombinant Cyn d 1 was expressed in Escherichia coli and yeast. The expressed proteins were analyzed by Western blotting to assess binding to Cyn d 1-specific monoclonal antibodies and IgE from sera of patients allergic to Bermuda grass pollen. RESULTS Two isoforms of Cyn d 1 were cloned. Recombinant Cyn d 1 expressed in bacteria bound two monoclonal antibodies raised against Cyn d 1 but was not recognized by IgE from sera of patients allergic to Bermuda grass pollen. Cyn d 1 expressed in yeast bound both the monoclonal antibodies and human IgE. CONCLUSION An IgE-reactive Cyn d 1 was expressed in yeast but not in bacteria, suggesting that posttranslational modifications (e.g., glycosylation), which occur in eukaryotic cells such as yeast, are necessary for the production of a biologically active allergen.

Cloning of a cDNA encoding a group-V (group-IX) allergen isoform from rye-grass pollen that demonstrates specific antigenic immunoreactivity

We have isolated and characterized the cDNA clone, 19R, that encodes an isoform of a major rye-grass pollen allergen, Lol p V [previously referred to as Lol p 1b; Singh et al., Proc. Natl. Acad. Sci. USA 88 (1991) 1384-1388; and Lol p IX; Suphioglu et al., Lancet 339 (1992) 569-572]. Clone 19R was isolated from a rye-grass pollen cDNA expression library using grass pollen-specific immunoglobulin E (IgE) antibodies (Ab) from an allergic serum pool. The nucleotide (nt) sequence of clone 19R potentially encodes a 33.8-kDa protein of 339 amino acids (aa). It possesses a leader peptide essentially identical to the previously characterized isoform of Lol p V (Lol p VA). This indicates a mature processed 31.3-kDa protein of 314 aa, correlating well with the size of the polypeptides revealed by Western analysis of pollen proteins using IgE Ab affinity purified from recombinant fusion protein (reFP) encoded by clone 19R as solid matrix. There is no N-glycosylation motif. The protein encoded by clone 19R, designated Lol p VB, has 66.4% identity and 80.4% similarity with Lol p VA. However, a Lol p VA-specific monoclonal Ab, FMC A7, does not recognize reFP encoded by clone 19R, indicating that Lol p VB does not share this epitope. Cross-reactivity studies using affinity purified IgE Ab showed that both isoforms share similar allergenic epitopes. Immunoblot analysis using sera from a population of 30 patients showed that 80% possess IgE Ab that recognize both Lol p V isoforms. Variation occurred in the signal intensities of IgE binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative cytology of the sperm cells of Brassica campestris and B. oleracea

Pollen grains of Brassica campestris L. var. acephala DC and B. oleracea L. were serially sectioned and examined using transmission electron microscopy to determine the three-dimensional organization of sperm cells within the microgametophyte and the quantity of membrane-bound organelles occurring within each cell. Sperm cells occur in pairs within each pollen grain, but are dimorphic, differing in size, morphology and mitochondrial content. The larger of the two sperm cells (Svn) is distinguished by the presence of a blunt evagination, which in B. oleracea wraps around and lies within shallow furrows on the vegetative nucleus and in B. campestris can penetrate through internal enclaves of the vegetative nucleus. This sperm cell contains more mitochondria in both species than the second sperm cell (Sua). This latter cell is linked to the first by a common cell junction with the Svn, but is not associated with the vegetative nucleus and lacks a cellular evagination. Such differences are indicative of a system of cytoplasmic heterospermy in which sperm cells possess significantly different quantities of mitochondria.

Molecular basis of IgE-recognition of Lol p 5, a major allergen of rye-grass pollen.

Grass pollen, especially of rye-grass (Lolium perenne). represents an important cause of type I allergy. Identification of IgE-binding (allergenic) epitopes of major grass pollen allergens is essential for understanding the molecular basis of interaction between allergens and human IgE antibodies and therefore facilitates the devising of safer and more effective diagnostic and immunotherapy reagents. The aim of this study was to identify the allergenic epitopes of Lol p 5, a major allergen of rye-grass pollen, immunodissect these epitopes further so that the amino acid residues critical for antibody binding can be determined and investigate the conservation and nature of these epitopes within the context of the natural grass pollen allergens. Peptides, 12-13 amino acid residues long and overlapping each other by 4 amino acid residues, based on the entire deduced amino acid sequence of the coding region of Lol p 5, were synthesised and assayed for IgE-binding. Two strong IgE-binding epitopes (Lol p 5 (49-60) and (265-276), referred to as peptides 7 and 34, respectively) were identified. These epitopes were further resolved by truncated peptides and amino acid replacement studies and the amino acid residues critical for IgE-binding determined (Lol p 5 (49-60) residue Lys57 and (265-276) residue Lys275). Sequences of these epitopes were conserved in related allergens and may form the conserved allergenic domains responsible for the cross-reactivity observed between pollen allergens of taxonomically related grasses. Furthermore, due to its strong IgE-reactivity, synthetic peptide Lol p 5 (265-276) was used to affinity-purify specific IgE antibodies which recognised proteins of other clinically important grass pollens. further indicating presence of allergenic cross-reactivity at the level of allergenic epitope. Moreover, Lol p 5 (265 276) demonstrated a strong capacity to inhibit IgE-binding to natural rye-grass pollen proteins highlighting the antibody accessibility to these sequences within the context of the natural allergens. Strong IgE-binding epitopes of Lol p 5 have been identified down to single critical amino acid residues and are shown to occur as linear or continuous domains in the natural conformation of natural Lol p 5 and other group 5 grass pollen allergens. The fact that such an allergenic synthetic epitope has the capacity to strongly inhibit IgE-binding to natural allergens highlight its potential for use as a candidate in future therapeutics to treat pollen-associated allergies.

Comparative analysis of fatty acids in pollen and seed of rapeseed

Abstract Lipids accounted for 31.7% of the dry wt of mature, viable pollen of rapeseed ( Brassica napus ). External lipids of the pollen coat, specified by the diploid parental genome, and internal cytoplasmic lipids, presumably specified by the haploid pollen genome, were separated by differential extraction. External lipids and paraffins represented 9.8 % of pollen dry wt, and contained predominantly linolenic (18:3) with significant amounts of palmitic (16:0), stearic (18:0) and myristic (14:0) acids, as well as paraffins. Internal lipids accounted for 21.9% of pollen dry wt and comprised predominantly 18:3 and 16:0. Mature seeds of B. napus comprised 45.0 % lipid, with a fatty acid composition different from that of either pollen fraction. Decanoic (10:0), lauric (12:0) and hexadecatrienoic (16:3) acids were detected in pollen but not in seeds. These data are discussed in relation to pollen biochemistry and the potential for pollen selection in rapeseed breeding.