Ubiquitin-specific proteases (UBPs) are a family of unique hydrolases that specifically

Ubiquitin-specific proteases (UBPs) are a family of unique hydrolases that specifically remove polypeptides covalently linked via peptide or isopeptide bonds to the C-terminal glycine of ubiquitin. However, the single Jasmonic acid manufacture and double homozygous plants are hypersensitive to the amino acid analog canavanine (CAN), supporting a role for these UBPs in particular, and the ubiquitin/26S proteasome pathway in general in aberrant protein turnover in plants. RESULTS Identification of UBPs in Arabidopsis Sullivan et al. (1990) first reported that plants have UBP-like activities capable of cleaving ubiquitin attached to other proteins via peptide or isopeptide linkages. To identify the responsible enzymes, we used the sequence of yeast UBP4 (Papa and Hochstrasser, 1993) as the query to search the Arabidopsis expressed sequence tag (EST) database for related proteins. Various yeast and Arabidopsis UBP sequences subsequently were used to examine the Arabidopsis bacteria artificial chromosome (BAC) and EST databases for additional candidate genes. This extensive search (last completed on September 26, 2000) ultimately identified 27 distinct genes that encode proteins with both the Cys- and His-box signature motifs (Wilkinson, 1997). Three of these Arabidopsis genes (family members (at least 24 of the 27) are actively expressed. By comparing the genomic sequences Jasmonic acid manufacture with their corresponding cDNAs, or by deducing intron/exon boundaries using alignments with possible paralogs, the complete coding regions were predicted for all 27 (Chandler et al., 1997; Rao-Naik et al., 2000; data not shown). In many cases, these coding sequences disagreed with those annotated in the AGI database. Figure ?Figure22 shows the organization of the genes was tentatively clustered into 14 subfamilies. In all cases, these four criteria were in agreement, supporting our subfamily classification (Figs. ?(Figs.22 and ?and3;3; data not shown). Percent amino acid sequence similarity among members of the predicted subfamilies ranged from 95% (for the subfamily, and the subfamily that contain five members each. The remaining seven subfamilies contain only a single gene (and or as the probe. As can be seen in Figure ?Figure4,4, only and are the only members in this subfamily. By sequence analysis of genomic and cDNA clones, the partial organization for and the complete organization of was determined (Fig. ?(Fig.5A).5A). Each contains a positionally conserved intron between the Rabbit polyclonal to TSP1 sequences for the F and His boxes, whereas is predicted to contain a second intron following the sequence for the G box. A 531-bp intron was detected upstream of the Met start codon in but could not be identified without an available cDNA sequence in that region. Figure 4 DNA gel-blot analysis of and from WT Arabidopsis (WT) and mutant plants. Arabidopsis genomic DNA was isolated from the ecotype WS and the double homozygous line, digested with and genes. A, Structure of and genes. Lines indicate introns and boxes indicate exons; white boxes, untranslated regions; gray/black boxes, translated regions. The Cys, … The encoded (Fig. ?(Fig.6A).6A). The activity of and and were identified that contain a T-DNA insertion in the coding region, 703- and 2,539-bp downstream of the respective translation start site, with the T-DNA either upstream of the Cys box (and lines were crossed and a double homozygote was isolated. To confirm that the and genes were affected in the lines, genomic DNA was isolated from the homozygotes and analyzed by DNA gel-blot analysis. In each case, the banding patterns of the mutant differed as predicted from that of WT at the respective loci (Fig. ?(Fig.4B;4B; data not shown). By using RT-PCR, we found that the T-DNA insertion also affected expression of the and genes. Whereas, the and mRNAs could be easily detected by RT-PCR, using as a template Jasmonic acid manufacture RNA isolated from WT plants treated with or without CAN, none could be detected using RNA from the corresponding mutants treated similarly (Fig. ?(Fig.7).7). As a result, we consider it likely that and represent null alleles. Figure 7 and mRNAs are absent in the Arabidopsis and seedlings grown for 6 d on media without CAN followed by an additional 2 d on media with or without … To assess the phenotypic functions of the and mutant plants were.