The Arabidopsis mutant pho1 is deficient in the transfer of Pi from root epidermal and cortical cells to the xylem. The PHO1 gene was identified by a map-based cloning strategy. The N-terminal half of PHO1 is mainly hydrophilic, whereas the C-terminal half has six potential membrane-spanning domains. PHO1 shows no homology with any characterized solute transporter, including the family of H+-Pi cotransporters identified in plants and fungi. PHO1 shows highest homology with the Rcm1 mammalian receptor for xenotropic murine leukemia retroviruses and with the Saccharomyces cerevisiae Syg1 protein involved in the mating pheromone signal transduction pathway. PHO1 is expressed predominantly in the roots and is upregulated weakly under Pi stress. Studies with PHO1 promoter-beta-glucuronidase constructs reveal predominant expression of the PHO1 promoter in the stelar cells of the root and the lower part of the hypocotyl. There also is beta-glucuronidase staining of enclodermal cells that are adjacent to the protoxylem vessels. The Arabidopsis genome contains 10 additional genes showing homology with PHO1. Thus, PHO1 defines a novel class of proteins involved in ion transport in plants.

The Arabidopsis mutant pho1 is deficient in the transfer of Pi from root epidermal and cortical cells to the xylem. The PHO1 gene was identified by a map-based cloning strategy. The N-terminal half of PHO1 is mainly hydrophilic, whereas the C-terminal half has six potential membrane-spanning domains. PHO1 shows no homology with any characterized solute transporter, including the family of H+-Pi cotransporters identified in plants and fungi. PHO1 shows highest homology with the Rcm1 mammalian receptor for xenotropic murine leukemia retroviruses and with the Saccharomyces cerevisiae Syg1 protein involved in the mating pheromone signal transduction pathway. PHO1 is expressed predominantly in the roots and is upregulated weakly under Pi stress. Studies with PHO1 promoter–β-glucuronidase constructs reveal predominant expression of the PHO1 promoter in the stelar cells of the root and the lower part of the hypocotyl. There also is β-glucuronidase staining of endodermal cells that are adjacent to the protoxylem vessels. The Arabidopsis genome contains 10 additional genes showing homology with PHO1. Thus, PHO1 defines a novel class of proteins involved in ion transport in plants.

A novel nodule-specific gene, LjNOD70, associated with late stages in Lotus japonicus nodule development and/or functioning was characterized. The LjNOD70 gene is a member of a small family of closely related L. japonicus genes. Two major mRNA species corresponding to the LjNOD70 gene were identified in nodules and shown to be the result of a mechanism resembling alternative splicing. The longer, presumably unspliced, mRNA species was shown to contain a single open reading frame (ORF), encoding a polytopic hydrophobic protein, LjN70, with a predicted molecular mass of 70 kDa. The second, presumably spliced, mRNA species was shown to be less abundant in nodules. The absence of the presumptive 'intron' was found to divide the reading frame into an upstream and a downstream ORF encoding the partial N- and C-terminal regions of the LjN70 protein, respectively. The predicted amino acid sequence of nodulin LjN70 revealed structural features characteristic of transport proteins, and was found to share similarity with the oxalate/formate exchange protein of Oxalobacter formigenes. Therefore, we postulate that the L. japonicus LjNOD70 gene family encodes nodule-specific transport proteins, which may have evolved as a result of exon-intron shuffling.

A detailed microscopical analysis of the morphological features that distinguish different developmental stages of nodule organogenesis in wild-type Lotus japonicus ecotype Gifu B-129-S9 plants was performed, to provide the necessary framework for the evaluation of altered phenotypes of L. japonicus symbiotic mutants, Subsequently, chemical ethyl methanesulfonate (EMS) mutagenesis of L. japonicus was carried out. The analysis of approximately 3,000 M(1) plants and their progeny yielded 20 stable L. japonicus symbiotic variants, consisting of at least 14 different symbiosis-associated loci or complementation groups. Moreover, a mutation affecting L. japonicus root development was identified that also conferred a hypernodulation response when a line carrying the corresponding allele (LjEMS102) was inoculated with rhizobia, The phenotype of the LjEMS102 line was characterized by the presence of nodule structures covering almost the entire root length (Nod(++)), and by a concomitant inhibition of both root and stem growth, A mutation in a single nuclear gene was shown to be responsible for both root and symbiotic phenotypes observed in the L, japonicus LjEMS102, line, suggesting that (a) common mechanism(s) regulating root development and nodule formation exists in legumes.

Crude extract was prepared from the sea anemone, Bunodosoma cavernata. The protein content of the extract was estimated to be 0.52 mg protein/ml. The extract was standardized based on the percentage inhibition of histamine-induced contraction of the guinea pig ileum, to determine the biological unit of activity (AU) of the extract. As extracts prepared on different occasions lost potency on storage, the stability of the extract was also investigated. Extracts prepared from fresh animals were about 15% more potent than those prepared from freeze-dried animals. However, freeze-dried animal extracts maintained their potency for about 6 months under storage at −20°C. Lethality studies gave an ld50 of 40 μg protein/kg mice i.p. Also, the crude extract dose-dependently hemolyzed human erythrocytes at room temperature. This activity was favoured by higher temperatures, which peaked at about 60°C, and by pH in the alkaline range. We conclude that the crude extract from B. cavernata, though highly toxic, may also contain some biologically active agents which include a haemolytic factor and antihistamine(s), as indicated by its histamine-blocking action.

A range of novel expressed sequence tags (ESTs) associated with late developmental events during nodule organogenesis in the legume Lotus japonicus were identified using mRNA differential display; 110 differentially displayed polymerase chain reaction products were cloned and analyzed. Of 88 unique cDNAs obtained, 22 shared significant homology to DNA/protein sequences in the respective databases. This group comprises, among others, a nodule-specific homolog of protein phosphatase 2C, a peptide transporter protein, and a nodule-specific form of cytochrome P450. RNA gel-blot analysis of 16 differentially displayed ESTs confirmed their nodule-specific expression pattern. The kinetics of mRNA accumulation of the majority of the ESTs analyzed were found to resemble the expression pattern observed for the L. japonicus leghemoglobin gene. These results indicate that the newly isolated molecular markers correspond to genes induced during late developmental stages of L. japonicus nodule organogenesis and provide important, novel tools for the study of nodulation.

Anchoring of proteins to membranes by glycosylphosphatidylinositols (GPIs) is ubiquitous among all eukaryotes and heavily used by parasitic protozoa. GPI is synthesized and transferred en bloc to form GPI-anchored proteins. The key enzyme in this process is a putative GPI:protein transamidase that would cleave a peptide bond near the COOH terminus of the protein and attach the GPI by an amide linkage. We have identified a gene, GAA1, encoding an essential ER protein required for GPI anchoring. gaal mutant cells synthesize the complete GPI anchor precursor at nonpermissive temperatures, but do not attach it to proteins. Overexpression of GAA1 improves the ability of cells to attach anchors to a GPI-anchored protein with a mutant anchor attachment site. Therefore, Gaa1p is required for a terminal step of GPI anchor attachment and could be part of the putative GPI:protein transamidase.