The maize regulatory protein Opaque-2 (O2) localizes to the nucleus in both maize and tobacco cells. Here we show that in-frame carboxy- and amino-terminal fusions of O2 to reporter protein beta-glucuronidase (GUS) were sufficient to direct GUS to the nucleus in transgenic tobacco plants and in transiently transformed onion cells. Two independent regions of O2 containing 135 and 149 amino acids were identified that were able to redirect GUS to the nucleus in both systems. A quantitative biochemical analysis of GUS in nuclei isolated from transgenic tobacco plants revealed that the second region was more efficient than the first one. The precise location of nuclear localization signals (NLSs) was determined using an onion transformation system. The first NLS was located between residues 101 and 135 and had the structure of a simian virus 40 NLS. The second NLS was located in the basic, DNA binding domain (between residues 223 and 254) and had a bipartite structure. The presence of one of the O2 NLSs in the basic domain is in complete agreement with similar findings of NLSs in the basic domain of three other basic/leucine zipper proteins, suggesting that this domain may be bifunctional. The effect of amino- versus carboxy-terminal GUS fusions is discussed.

The maize regulatory protein Opaque-2 (02) localizes to the nucleus in both maize and tobacco cells. Here we show that in-frame carboxy-and amino-terminal fusions of 0 2 to reporter protein p-glucuronidase (GUS) were sufficient to direct GUS to the nucleus in transgenic tobacco plants and in transiently transformed onion cells. Two independent regions of 0 2 containing 135 and 149 amino acids were identified that were able to redirect GUS to the nucleus in both systems. A quantitative biochemical analysis of GUS in nuclei isolated from transgenic tobacco plants revealed that the second region was more efficient than the first one. The precise location of nuclear localization signals (NLSs) was determined using an onion transformation system. The first NLS was located between residues 101 and 135 and had the structure of a simian virus 40 NLS. The second NLS was located in the basic, DNA binding domain (between residues 223 and 254) and had a bipartite structure. The presence of one of the 0 2 NLSs in the basic domain is in complete agreement with similar findings of NLSs in the basic domain of three other basiclleucine zipper proteins, suggesting that this domain may be bifunctional. The effect of amino-versus carboxy-terminal GUS fusions is discussed.

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The molecular basis for the low level expression of three alleles of the maize waxy (Wx) gene has been described. Each allele contains a retrotransposon in intron sequences. These insertions represent previously undescribed elements, and their association with three wx alleles indicates that retrotransposon elements are important agents of spontaneous mutation in maize. For each allele, element sequences are spliced from pre-mRNA with the surrounding intron even though the insertions increase intron length by approximately 40- to 60-fold. In addition, despite differences in element sequences, insertion sites, and relative orientations, each element disrupts long-range splice site recognition leading to novel Wx transcripts where exons both upstream and downstream of the insertion site are skipped. The expression of wx alleles with large insertions in introns provides support for studies that indicate that the primary cis requirement for maize introns is the splice donor and acceptor sites.

The molecular basis for the low level expression of three alleles of the maize waxy (Wx) gene has been described. Each allele contains a retrotransposon in intron sequences. These insertions represent previously undescribed elements, and their association with three wx alleles indicates that retrotransposon elements are important agents of spontaneous mutation in maize. For each allele, element sequences are spliced from pre-mRNA with the surrounding intron even though the insertions increase intron length by approximately 40- to 60-fold. In addition, despite differences in element sequences, insertion sites, and relative orientations, each element disrupts long-range splice site recognition leading to novel Wx transcripts where exons both upstream and downstream of the insertion site are skipped. The expression of wx alleles with large insertions in introns provides support for studies that indicate that the primary cis requirement for maize introns is the splice donor and acceptor sites.