The structure and expression of a maize ubiquitin fusion gene --Keqin Chen, David A. Somers, and Irwin Rubenstein Ubiquitin fusion genes encode a hybrid protein in which ubiquitin is fused at its carboxyl terminus to a tail sequence of either about 50 or 80 amino acid residues. Similar ubiquitin fusion proteins have been found in yeast, humans, and plants. The amino acid sequence of the tail region is highly conserved among all eukaryotes investigated. The tail sequence is unrelated to the ubiquitin sequence and contains a putative metal-binding, nucleic acid-binding domain (Cys-X2-4-Cys-X2-15-Cys-X2-4-Cys). Recent research indicates that these short tail polypeptides are located in ribosomes and may play a role in ribosome biogenesis. We have isolated a maize ubiquitin fusion gene and determined its structure. We have also studied the expression of this fusion gene in maize tissues.

An ubiquitin fusion gene clone was identified by screening a lambda maize W22 genomic library with a short oligonucleotide probe derived from the sequence for a yeast ubiquitin fusion gene. A 5 kb BamHI fragment containing the ubiquitin fusion gene sequence was subcloned into the pUC119 vector. The sequence of the gene has been determined; it contains no introns. The fusion gene consists of a ubiquitin monomer sequence (228 bp long) and an extension sequence (234 bp long). The deduced amino acid sequence of the ubiquitin portion is identical to the sequence of the maize ubiquitins we have previously studied. The tail extension sequence consists of 78 residues and is highly homologous to the sequence found in yeast, human, and Arabidopsis (Fig. 1).

(76 maize ubiquitin amino acid residues)-A-K-K-R-K-K-K-E-Y-T-K-P-K-K-I-K-H-K-H-K-K-V-K-L-A-V-L-Q-F-Y-K-V-D-D-A-T-G-K-V-P-A-S-A-R-
C-P-N-T-E-C-G-A-G-V-F-M-A-N-H-F-D-R-H-Y-C-G-K-C-G-L-T-Y-V-Y-N-Q-K-A

Figure 1. Deduced amino acid sequence of a maize ubiquitin fusion protein using the standard one-letter code to denote the amino acid residues: A = Alanine, R = Arginine, N = Asparagine, D = Aspartic Acid, C = Cysteine, Q = Glutamine, E = Glutamic Acid, G = Glycine, H = Histidine, I = Isoleucine, L = Leucine, K = Lysine, M = Methionine, F = Phenylalanine, P = Proline, S = Serine, T = Threonine, W = Tryptophane. Y = Tyrosine, and V = Valine.

The gene is transcribed as a 900-nucleotide transcript. Northern blots indicate that the gene is expressed at various levels in different maize tissues. For example, it is expressed at high levels in endosperm tissue (after pollination) and in the coleoptile tissue of young seedlings. The expression of the fusion gene appears to be regulated during endosperm development. Genomic Southern blots of maize inbred W22 DNA demonstrate that fusion gene sequences are present in more than one copy in the maize genome.

The promotor region of the fusion gene has been characterized by sequence analysis and S1 mapping of the transcription initiation site. An 800 nucleotide fragment of the promotor sequence has been fused to a GUS reporter gene to study the regulation of the fusion gene's expression. This DNA was delivered directly into Black Mexican suspension culture cells by means of DNA-coated gold particles accelerated by the high-voltage-induced explosion of a water droplet (McCabe et al., Bio\Technology 6:923, 1988). The GUS gene under the control of the fusion gene promotor has expressed.

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