Studies on the tandemly duplicated 27kD zein genes had identified a mitotic DNA rearrangement at this locus in particular stocks of A188 (Das et al., PNAS 87:7809, 1990). This appeared to be a two-step process, consisting of a homologous recombination between the repeats of the duplicated S allele, and a second rearrangement at the 5' end (Fig. 1). Cloning the rearranged allele has now shown that the second event is the insertion of a 7.3kb element 1.1kb upstream of the A gene (Fig. 1). By using PCR primers near the insertion site, we have shown that this insertion is only found in the Ra allele which has only the A copy, and not in the S allele which has both the A and B copies of the duplication (Fig. 1). The Ra allele is found in many other inbred lines, e.g. W64A, B37 and A619.

We have characterized the 7.3kb insertion element which we designate Zeon1 ( zein retrotransposon) because it has many of the characteristics of a retrotransposon. Its insertion site is flanked by a typical 5bp target site duplication (Fig. 1). In addition, a second imperfect duplication is present at one end (Fig. 1). Two LTRs (long terminal repeats) of 649bp and 662bp have the typical structure of U3-R-U5, including nucleotide sequences similar to a maize TATA box and a poly-A signal. Zeon1 also has a PBS (primer binding site) and a purine-rich sequence at the 5' and 3' ends of the internal sequence. Its PBS consists of ten nucleotides complementary to the 3' end of Lys-tRNA; most plant retrotransposons have a Met-tRNA binding site. The internal sequence of Zeon1 has several open reading frames. The largest one is a gag-related ORF of 375 aa, that includes a putative zinc finger in the nucleic acid binding domain. Comparison of the zinc finger region of Zeon1 to that of other retrotransposons and retroviruses shows that Zeon1 has one extra amino acid (Fig. 2). The lengths of the other longest ORFs are 92 aa and 113 aa, but none of them shows significant homology to reverse transcriptases.

Using probes for the LTR and for an internal sequence, we estimate a copy number of around 1000 for LTR-related sequences, and between 300-400 for the internal sequence. Sequence comparisons show two regions of 91bp and 120bp in the LTR of Zeon1 that are homologous to the maize retroelement Cin1 (Shepherd et al., Nature 307:185, 1984). Sequences homologous to the LTR are also found at the 3' flanking region of a 19kD zein gene (Kriz et al., MGG 207:90, 1987).

Figure 1. The upper panel shows the restriction maps of S and Ra alleles. The A and B copies of the duplications of the S allele are drawn on separate lines to highlight homology. Homologous regions are represented by the heavy line, open boxes indicate coding sequences and the striped box represents an insertion unique to the B copy. The crosshatched box separates the duplications. The Ra allele has a 7.3kb insertion, Zeon1, located 1.1kb upstream of the coding sequence. In addition, its formation requires a second homologous crossover between the repeats at the indicated site. Transcription direction is from left to right. Restriction site key: B = BglII, C = ScaI, E = EcoRI, H = HindIII, L = SalI, P = PstI, S = SacI, X = XbaI. The lower panel shows nucleotide sequences at the Zeon1 insertion site in the S and Ra alleles. Note the 5bp target site duplication, the 8bp terminal inverted repeats flanking Zeon1, and the imperfect 6bp duplication immediately adjacent to the insertion.

Figure 2. A comparison of the gag-related ORF of Zeon1 with the conserved nucleic acid binding domain found in retroviruses and retrotransposons is shown. Amino acid sequences are indicated in capital letters, and the corresponding nucleotide sequence for Zeon1 is indicated in lower case. Tnt1 is a retrotransposon from Nicotiana tabacum; Copia is a retrotransposon from Drosophila melanogaster; MMULV is the Moloney murine leukemia virus; RSV is the Rous sarcoma virus; HIV-1 is the human immunodeficiency virus. The conserved cysteine, histidine and glycine residues involved in the zinc-binding domain are marked.

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