Sequence analysis of an opaque2 mutant of Zea mays
--B. Lazzari, P. Ciceri, F. Cellini and A. Viotti

The opaque2 mutant of the Bianchi o2 maize line, recovered as a spontaneous mutation in the early sixties (Bianchi, personal communication), previously analysed at the genetic level (Nelson, Maydica 12:81-96, 1967; Salamini, Cold Spring Harbor Symp. Quant. Biol. 45:467-476, 1980) and more recently investigated at the molecular level, has been introgressed in four different lines: W22 and A69Y (Istituto Sperimentale per la Cerealicoltura, Bergamo, Italy), NYR and 3316 (Dipartimento di Genetica e Biologia dei Microorganismi, Milan, Italy). Southern and Northern analysis of these different lines reveals no difference in regard to o2 gene and transcript patterns. It should be remembered that this o2 allele produces a transcript normal in size and similar in level in respect to the wildtype line (Dolfini et al., Dev. Genet. 13:264-276, 1992). In order to obtain the cDNA clone of this allele we synthesised four oligonucleotides: one in the leader sequence of the O2 wildtype allele, one in the trailer sequence, and two internal to the coding region (Schmidt et al., PNAS 87:46-50, 1990). Specific amplification of the two regions of the o2 allele has been carried out by PCR on total RNA extracted from 20 DAP (days after pollination) endosperms of the A69Yo2 maize line and reverse transcribed using oligo-dT primer and MoMuLV reverse transcriptase. The amplified sequences were treated with the large fragment of DNA polymerase I (Klenow fragment) and cloned in the pBSKS vector (Stratagene). We obtained two clones that represent the whole cDNA sequence of the o2 Bianchi allele, named o2-Italian, one spanning from about 100 bp before the ATG codon to base 936 of the coding sequence (after the leucine zipper motif), and the second from base 667 of the coding sequence (before the basic domain) to some 40 bases after the stop codon. More copies of the two clones have been prepared, coming from different amplification reactions, in order to verify the reliability of the method. All these clones have been sequenced and compared, showing no significant difference among the nucleotide sequences. However, comparison of the deduced amino acid sequence of o2-Italian with the O2 wildtype sequences of both A69Y and W22 maize lines revealed various differences. The most important difference is a deletion of a sequence of 7 amino acids in the basic domain.

As the basic domain of the O2-bZip transcriptional factor is involved in DNA binding, these preliminary data suggest the possibility of a loss of function of the o2-Italian allele due to the lack of binding activity. This hypothesis is supported by the results of Southwestern experiments carried out using oligonucleotides containing the O2 target sequence as probes. Moreover, the deleted amino acids include the short RKRK sequence which constitutes the first part of the bipartite NLS (nuclear localising sequence) contained in the opaque2 basic domain (Varagona et al., Plant Cell 4:1213-1227, 1992). This could lead to lower efficiencies in transporting O2 to the nucleus, even if O2 contains another short low-efficiency NLS in the amino-terminal region. 

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