MILAN, ITALY
Istituto Biosintesi Vegetali-CNR; Centro Miglioramento Sanitario Colture Agrarie - CNR

Molecular characterization of o2-T: a mutant allele at the opaque2 locus
--B. Lazzari, P. Ciceri, R. Carzaniga, A. Genga, F. Faoro and A. Viotti

Several wild-type and mutant alleles of the opaque2 gene have been characterized at the DNA, RNA and protein levels (Bernard et al., Plant Mol. Biol. 24:949-959, 1994); one of them, referred to as o2-T and present in the W64A genetic background, has been further investigated by sequence analysis. This allele derives from the spontaneous mutation of the O2-wI allele of the W64A wild-type line. Both the wild-type and mutant alleles have been amplified by RT-PCR starting from total RNA of immature endosperm at 15 days after pollination. The amplified fragments have been cloned in pBluescript (Stratagene) and sequenced.

The sequence analysis of these two clones shows 99% homology in the 5' end of the sequences, but a deletion in the mutated allele causes a frame shift which leads to a stop codon, in accordance with data obtained by sequence analysis of the corresponding genomic regions of both W64A+ and o2-T. The deduced truncated protein has a molecular mass of 24kD and an isoelectric point of 4.37 (data obtained by computer analysis). This protein could correspond to the 40 kD polypeptide detected by Western blot analysis, as the real and apparent MW of the O2 protein on denaturing gels do not correspond, due to the presence of particular amino acid sequences in the N-terminus of the O2 polypeptides which could alter the normal correlation between the relative mobility and the molecular mass (Bernard et al., Plant Mol. Biol. 24:949-959, 1994; Liang et al., Electrophoresis 13:346-353, 1992). The predicted isoelectric point is in accordance with experimental data, as the o2-T polypeptide analyzed by IEF migrates as a single band with a pI of about 4.3.

Electron microscopy examination of immunogold labeled sections of maize endosperm shows that the o2-T polypeptide is located in the cytoplasm, while the wild-type is 90% in the nucleus. It is important to note that the truncated protein lacks the basic domain structure that, in the case of Opaque-2 protein, has a double function of DNA binding and nuclear localization. The basic domain, in fact, contains the NLS B bipartite structure, which seems to be the major factor responsible for Opaque-2 nuclear targeting. Another targeting signal, NLS A, is present in Opaque-2 and o2-T proteins, but seems to be less efficient than NLS B in redirecting the polypeptide to the nucleus (Varagona et al., Plant J. 5:207-214, 1994). 


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