Characterization and expression of genes encoding different polyubiquitin mRNAs
--Ling Liu, David B. Walden and Burr G. Atkinson

Ubiquitin is a remarkably conserved 76-amino acid-containing protein found in all eukaryotic cells examined. In eukaryotes, ubiquitin is encoded by a multigene family and the active ubiquitin monomer originates from post-translational cleavage of two different forms of a polyprotein precursor. One form of the precursor consists of a number of direct repeats of ubiquitin (i.e. polyubiquitin), and the other form consists of monoubiquitin linked via its C-terminus to a small, unrelated protein.

In this communication, we characterize the structure and expression of the sequences in genomic and cDNA clones encoding different polyubiquitin genes in an inbred line (0h43). One of the clones, g/cMub1, encodes an mRNA transcript containing seven tandem repeats of a nucleotide sequence for a ubiquitin monomer; the last repeat contains an extra, glutamine-encoding codon before the stop codon. The 3' UTR is 190 nucleotides long and contains 2 partial polyadenylation signals located 78 and 22 nucleotides upstream from the polyadenylated site. The gene for this polyubiquitin contains a 1005 nucleotide intron which, like one found in the sunflower (Binet et al., Plant Mol. Biol. 17:395-407, 1991), has its 3' splice site just before its ATG start codon. Another clone, g/cMub9, encodes an mRNA transcript containing five tandem repeats of a ubiquitin-encoding sequence; in this case, the extra codon in the last repeat encodes tyrosine. The 3' UTR of g/cMub9 consists of 206 nucleotides and contains a complete, animal-like polyadenylation signal 20 nucleotides upstream from the polyadenylated site. Since, at this time, the cDNA we have for this gene lacks a 5' UTR, we are unable to document whether or not its 5' UTR is also interrupted by an intron.

We have characterized the expression of these genes in radicles and plumules from control (25 C) and heat-shocked (42.5 C) 5-day-old seedlings by Northern and dot blot hybridization analyses of poly(A)+ RNAs isolated from the total cellular RNAs and from the polyribosomal RNAs. The nucleotide sequences used for these analyses included a sequence from the ORF of g/cMub1 (scMubC1-1; a 0.473kb XhoI/SacI-excised DNA fragment) common to both genes, and a sequence from the 3' UTR of each gene which is specific for each gene (MubC1-1, a 0.204kb PCR-generated DNA fragment specific for g/cMubC1; and scMubC9-3, a 0.224kb AccII/EcoRI-excised DNA fragment specific for g/cMubC9). Results from the Northern and dot blot hybridization analyses establish that the mRNA transcripts corresponding to the sequence in g/cMubC1 is up-regulated in both the total RNA and polyribosomal RNAs during heat shock, while the level of the mRNA transcript corresponding to g/cMubC9 is not affected in either case by heat shock. These differences in the expression of these genes in response to heat shock clearly indicate the need for using gene-specific probes when assessing the expression of mRNA transcripts originating from different members of this multigene family. 

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