Isolation and Characterization of a Rice Full-Length cDNA Clone Encoding a Polyubiquitin

Ubiquitin is a highly conserved 76-residue protein found in both the nucleus and the cytoplasm of a11 eukaryotic cells (Finley and Varshavsky, 1985). The protein has been implicated in many cellular functions, including the selective targeting of cellular proteins for degradation, chromatin structure, and response to heat shock and other stresses (Monia et al., 1990). Many eukaryotic ubiquitin proteins and genes have been isolated and characterized (Callis and Vierstra, 1986; Schlesinger and Bond, 1987). The ubiquitin protein exists either as a free monoubiquitin or as a conjugated form of monoubiquitin linked via its carboxyl-terminal Gly residue to a Lys residue of a variety of proteins (Monia et al., 1990). Genes for ubiquitins are also known to exist as two types of natural gene fusions (Callis and Vierstra, 1986; Schlesinger and Bond, 1987; Monia et al., 1990). One type encodes monoubiquitin fused to unrelated polypeptides of either 52 or 76 to 80 amino acids. These fused genes were shown to be ribosomal protein genes in yeast (Monia et al., 1990), rice (Nishi et al., 1993), and severa1 other plants (Callis and Vierstra, 1986). The other type of natural gene fusion consists of tandem head-to-tail repeats of 228 bp encoding polyubiquitin, and the number of repeats varies among genes and organisms from 5 in yeast to 12 in Xenopus. Posttranslational processing of these polyubiquitins is responsible for generating monomeric units of the protein, which in turn are used in ubiquitination of a battery of proteins (Monia et al., 1990). Ubiquitinated proteins may then be involved in many of the cellular functions mentioned above. We have screened approximately 2 X 104 plaques of a cDNA library prepared from etiolated rice seedling (provided by Dr. R. Wu) using a ubiquitin-specific oligonucleotide probe, 5 ’ G AC T AC A AC ATC C AG A AG G A G 3 ’ , derived from a conserved region of plant ubiquitin gene sequences (Table I). After secondary screening, 15 independent clones were identified that hybridized strongly to the oligonucleotide probe. Phage DNA was purified from these 15 plaques, digested with EcoRI, and analyzed by agarose gel electrophoresis and DNA blot hybridization using the oligonucleotide probe. As a result, we have isolated five ubiquitin cDNAs that are different in size and hybridized specifically to the oligonucleotide probe. One of these clones, called Ubql, was chosen to be sequenced. Nucleotide sequences from both strands of the Ubql cDNA were determined. Sequence analyTable 1. Characteristics of rice Ubq 1 cDNA