Primary structure of the soybean nodulin-35 gene encoding uricase II localized in the peroxisomes of uninfected cells of nodules

Abstract

, a subunit of nodule-specific uricase (uricase II) of soybean (Glycine max), is shown to be preferentially synthesized on free polysomes during nodule development and is localized in peroxisomes of the uninfected cells of this tissue. A cDNA clone, isolated by using mRNA from immunoprecipitated polysomes, revealed the primary structure of this protein with a molecular mass of 35,100. That this clone represents N-35 was confirmed by comparing the deduced amino acid sequence with the partial sequence of a CNBr-cleaved peptide of purified N-35. Southern blot hybridizations with genomic DNA suggest that there are several EcoRI fragments containing N-35 sequences. Three of these sequences were isolated from a genomic library of soybean. Nucleotide sequence analysis showed that the complete gene extends almost 5000 base pairs on two EcoRI fragments and the coding region (309 codons) is interrupted by seven introns ranging in size from 154 to 1341 base pairs. Lack of a signal sequence and its translation on free polysomes suggest that N-35 is posttranslationally transported to the peroxisomes. Furthermore, there is no cross-hybridization of N-35 cDNA with RNA from young (3-to 4-day) roots and leaves, indicating that the observed "uricase" activity in these tissues is due to the product of a different gene.Tropical legumes primarily transport fixed nitrogen as ureides, allantoin, and allantoic acid (refs. 1 and 2, see also ref.3). These compounds are formed in root nodules via de novo purine biosynthesis and oxidation. It has been suggested that the enzymes involved in purine biosynthesis are located in the proplastids (4), whereas the oxidation ofpurine occurs in microbodies (peroxisomes) (5-7). A number of enzymes involved in ureide metabolism have been found to be associated with peroxisomes (see refs. 7 and 8), which are more abundant in the uninfected cells of nodules (9). Recent clone from soybean and determined their structures at the nucleotide level. Of general interest are features that may be responsible for the specificity to peroxisomes of this gene product and those involved in tissue-specific induction following infection of a legume plant by Rhizobium. To our knowledge, isolation of a eukaryotic gene that codes for a peroxisomal protein has not been reported previously, and studies of its structure may be of significance in understanding the biogenesis and specialization of the peroxisomes (12, 13) for various metabolic reactions (7,8). Furthermore, comparison of its structure with those of other nodulins (14, 15) should allow identification of sequences that may be responsible for the temporal control in regulating the expression of this gene. MATERIALS AND METHODSPlant Tissues and Isolation of RNA and DNA. Soybean (Glycine max cv. Prize) seeds were inoculated with Rhizobium japonicum (strain 61A76) and germinated as described (16). Infection zone from the primary root of less than a week and nodules from 2-to 3-week-old plants were obtained. Fresh tissues were used for immunocytochemical stud...

Coordinate expression of transcriptionally regulated isocitrate lyase and malate synthase genes in Brassica napus L.

“…We did not detect extensive regions of amino acid sequence similarity between isocitrate lyase and other plant peroxisomal proteins, i.e. malate synthase (Comai et al, 1989), soybean uricase (Nguyen et al, 1985), and spinach glycolate oxidase (Volokita and Somerville, 1987). However, we note that the carboxyterminal three amino acids (Ser-Arg-Met) of isocitrate lyase are similar to those implicated to be both necessary and sufficient to target other proteins to peroxisomes (Ser-Lys/ His-Leu; Gould et al, 1988).…”

Purification, cDNA Cloning, and Developmental Expression of the Nodule-Specific Uricase from Phaseolus vulgaris L.

“…In (14). cDNA probes for Northern hybridization were prepared by nick-translation as described (12).…”

“…A 35,000 mol wt subunit of soybean root-nodule uricase (nodulin-35) has been purified to homogeneity; this enzyme has been 'Supported in part from Consejo Nacional shown to differ in physicochemical properties and to have no antigenic cross-reactivity with the enzyme found in uninfected roots or leaves (2). cDNA and genomic clones encoding for uricase II have been isolated and the deduced primary structure of the enzyme from DNA sequence has been reported (14). The lack of cross-hybridization of nodulin-35 (N-35) cDNA with RNAs from other tissues indicated that N-35 is a nodule-specific gene product which is induced only during the Rhizobiumsoybean symbiosis.…”

Appearance of Purine-Catabolizing Enzymes in Fix+ and Fix− Root Nodules on Soybean and Effect of Oxygen on the Expression of the Enzymes in Callus Tissue

“…Xanthine dehydrogenase is associated with discrete organelles and present exclusively in uninfected cells as demonstrated by Nguyen (20), while Triplett (28) found xanthine dehydrogenase activity in the infected cells. Uricase is present in peroxisomes in uninfected cells (21). In sucrose density gradient studies allantoinase was demonstrated to be associated with the microsomal fraction (11).…”