The b-32 protein of endosperm has been described as a putative regulatory factor for the synthesis of zeins, the major group of storage proteins. Recently, the cDNA coding for the b-32 protein has been cloned and the complete amino acid sequence of the protein derived (Di Fonzo et al., Mol. Gen. Genet. 212:481, 1988). The cloning and sequencing of three b-32 genes from two different inbred lines (W64A and A69Y) as well as the Southern analysis, demonstrate that the b-32 genes form a polymorphic gene family that, in the case of W64A, is constituted by at least three genes.

The two isolated W64A genes and the previously reported cDNA clone are extremely similar and most of the observed nucleotide variations account for an amino acid replacement or for an insertion/deletion of 1-2 residues within the amino acid sequence. The genes b-32.129 (W64A) and b-32.152 (A69Y) differ from the b-32.120 gene (W64A) and b-32.66 cDNA clone (W64A) in three 1-nucleotide insertions, covering the central part of the coding region. The reading frame changes three times within 219 nucleotides and, as a result, the translation of the central domain of the b-32 protein changes markedly from one to the other set of clones. This contrasts with the preservation in all cases of the N-terminal and C-terminal domains.

The central region of the b-32 protein is a highly hydrophilic sequence, very rich in acidic residues and, probably, poorly structured. These characteristics are also valid for the protein sequence that is derived from the b-32.120 gene. However, the amino acid sequences of the central domain deduced from the nucleotide sequence of the b-32.129 and b-32.152 genes display a very different character. In fact, the central sequence is less hydrophilic, contains a rather low proportion of acidic residues and an increased amount of basic amino acids.

All b-32 genes described appear to be functional genes, as they possess the typical characteristics for it. The presence in the developing endosperm cell of two types of b-32 proteins, having two alternative central domains, is of great interest for these molecules in relation to possible regulatory mechanisms in which they could be involved. In fact, it has been reported that acidic surfaces of regulatory proteins can interact with the chromatin of the promotor region causing a local relaxation and facilitating the expression of the gene. If this is the case for the b-32 protein, a molecule possibly involved in the regulation of zein genes, the simultaneous presence within the cell of two molecular species with an alternative central domain affords a possible mechanism of switching on and off via local chromatin relation, depending on which of the two molecules is present nearby the promotor region of a given gene.

The two b-32 genes isolated from the inbred line W64A are very similar with regard to the flanking sequences and they possess the same motifs that apparently are relevant for gene expression. Therefore, it seems likely that they are coordinately expressed, probably being regulated by the same transacting factors. In addition to the typical elements that participate in the overall activation of the transcriptional machinery (CATAGA and TATA boxes), a long stretch of DNA rich in A+T is present at about 410 nucleotides upstream of the first ATG codon. Two additional motifs are present, corresponding to a proximal inverted repeat (PIR) and to a far inverted repeat (FIR) flanking the A+T rich element. It is probable that some if not all of these motifs are cis-acting elements participating in the regulation of the b-32 genes.

A comparison between gene b-32.129 (W64A) and gene b-32.152 (A69Y) is of special interest. From the coding sequence they could be considered as polymorphic genes from two different inbred lines showing a high level of homology. However, the 5' flanking region upstream the CATAGA motif shows great divergence, since the b-32.152 gene possesses several duplications and long inverted repeats, when compared with the b-32.129 gene. To date, we have no further sequence 5' upstream region of the b-32.152 gene. However, our data indicate that an important polymorphic variation exists between the two genes that may correlate with a different regulatory mechanism for each case. From the above considerations, it is proposed that the b-32 genes represent a family of regulated regulatory genes that play a role as intermediate elements of an unknown regulatory chain. The fact that the same mutants (o2 and o6) control the expression of both b-32 and zein genes, makes it of interest to investigate a possible mechanism of gene regulation of zeins in which the b-32 genes and their protein products might be involved.

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