Maize endosperm prolamins, or zeins, fall into four major classes viz., a, b, g, and d.  Of these, zeins belonging to b- and d- classes contain 10% and 22% of methionine/mol protein, respectively.  Being an essential amino acid, methionine forms an important constituent in both human and animal diets.  Previously, the cloning of a 10 kDa high-methionine zein gene belonging to the d-class was reported by our laboratory (Kihara et al., Mol. Gen. Genet. 211:  477-484, 1988).  We report here the cloning and analysis of dzs23, which has 86% DNA sequence similarity to the 10 kDa structural gene (referred to from here on as dzs10).

The predicted zein encoded by dzs23 has a 21 aa long leader similar to that of DZS10 zein and the mature protein is 1.5 times longer than DZS10 (Fig. 1A). DZS23 is longer, due to an internal duplication thereby increasing its methionine content to 26% as compared to 22%/mol of DZS10. Surprisingly, the predicted DZS23 zein contains 1 lysine and 2 tryptophan residues. Presence of the lysine residue was confirmed by endoproteinase Lys-C digestion of DZS23 which released an 18 kDa peptide (data not shown). Both lysine and tryptophan are absent in the 10 kDa zein and normally underrepresented in other zeins.

Synthesis of a T7 Tag-DZS23 fusion protein allowed us to test the cross-reactivity of a polyclonal antibody directed against DZS10. The d-zein antibody cross-reacted to the in vitro synthesized DZS23 fusion protein (data not shown).  This result led us to study the levels of both DZS10 and DZS23 in various maize inbred lines (Fig. 1B).  Three patterns of d-zein levels were found.  Inbred Mo17 was low in both; BSSS-53 was low in DZS23 but very high in DZS10; B37 and A619 had moderately high levels of both d-zeins.  The antisera used as control to detect a-zeins also react with lower sensitivity to d-zeins.  Protein blot of zeins from mature kernels (Fig. 1B) and from in vitro synthesis (not shown) provided a size estimate of 23 kDa for the novel d-zein, thus the designation DZS23.

In order to simultaneously study the expression of both dzs10 and dzs23 in developing kernels, we resorted to using primer extension. An antisense oligo was synthesized which fit the following three criteria: (i) whose binding site was conserved between the two genes, (ii) which was ~ 100 bp from the transcription start site and (iii) whose extension products would show size polymorphism. The primer extended products corresponding to both d-zeins in four maize inbred lines is shown in Fig. 1B. The doublet corresponding to dzs23 transcript is possibly due to strong secondary structures at the 5'-end as predicted from its sequence. dzs23 RNA are detected in Mo17, no transcripts are detected by this analysis in BSSS-53. Identity of dzs23 transcript from Mo17 was confirmed by cloning and sequencing the RT PCR product. This analysis also revealed at least two polyadenylation sites in the mRNA. Based on its high methionine content as well as the presence of both lysine and tryptophan, DZS23 may be a good candidate for improving the nutritional quality of corn using the transformation technology.

Figure 1. Characterization of dzs23 gene.

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