Characterization and expression of genes encoding two different ubiquitin-fusion protein mRNAs
--Ling Liu, David B. Walden and Burr G. Atkinson

In this communication, we characterize the sequence and expression of two different ubiquitin fusion-protein genes, MubG7 and MubG10, in an inbred line (0h43). Both of the genes contain an ORF of 468 nucleotides which encodes for a polyprotein consisting of a ubiquitin monomer and an unrelated extension (fusion) protein consisting of 79 amino acids. While the ORFs of these genes share 95% identity, the first 300 nucleotides in the 5' and 3' flanking regions share only 30% and 46% identity, respectively. The derived amino acid sequence of the ubiquitin moieties encoded from MubG7 and MubG10 are identical to each other as well as to those derived from the maize polyubiquitin genes characterized elsewhere in this Newsletter (Liu et al.). The derived amino acid sequence of the fusion-proteins encoded from MubG7 and MubG10 differ by 5 amino acids. Both fusion-proteins are highly basic (pI ~10.8), and both contain sequence motifs for protein localization to the nucleus as well as zinc-finger structures for nucleic acid binding.

The expression of mRNA transcripts encoded from these genes was investigated in radicals and plumules from control (25 C) and heat-shocked (42.5 C) 5-day-old seedlings (0h43) by Northern and dot blot hybridization analyses. Poly(A)+ RNAs, isolated from the total cellular RNAs and from polyribosomal RNAs, were probed with a DNA fragment from the fusion-protein sequence which is common to both of these genes (scMubG10-C-2; a 0.323kb NarI/BglII-excised DNA fragment from MubG10 which shares 95% identity with fusion-protein sequence in MubG7). Results from the Northern and dot blot hybridization analyses, using poly(A)+ RNAs from the total cellular RNA, support the contention (Christensen and Quail, Plant Mol. Biol. 12:619-632, 1989) that the level of mRNA transcripts for these fusion-proteins does not change during heat shock. However, when poly(A)+ RNAs from the polyribosomes of these tissues were similarly analyzed, the results disclosed that there is a marked depression in the levels of the fusion-protein mRNA transcripts associated with the polyribosomes from tissues undergoing heat shock. These studies demonstrate that the genes encoding the maize ubiquitin fusion-proteins are not heat shock-inducible genes. Moreover, the decrease in the amount of their transcripts on polyribosomes during heat shock implies that the synthesis of these proteins is, in fact, depressed by heat shock. 

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