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-
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
to the MNL 64 On-Line Index
Return to the Maize Newsletter Index
Return to the Maize Genome Database Page