, 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...