University of Wisconsin

Analysis of a palindromic DNA sequence motif
--John C. Prostko and Tom Quayle

A recent analysis of the structure of zein genes and their flanking regions uncovered an unusual cluster of palindromic DNA sequences located at approximately two kilobase pairs upstream of a zein gene. Four nearly identical copies of this structure, each a GC-rich 17- to 21-base pair perfect palindrome, are contained in a stretch of only 142 base pairs. These four maize palindromic units (MPUs) are separated from each other by precise integral turns of the B-form DNA helix. Additionally, they are relatively abundant in the genome of maize, as shown by Southern blot analysis (Quayle et al. Gene 80:249-258, 1989) Furthermore, the MPUs are specifically bound by nuclear proteins in a tissue- and developmental-specific manner (Quayle, unpublished results).

A more accurate copy number determination and cloning of additional MPUs has been accomplished using a primary genomic library of maize in lambda phage EMBL3. We have recently screened this library via DNA-DNA hybridization on nitrocellulose membrane plaque lifts, and have isolated 32 MPU-containing phages. The minimum copy number for the MPUs was estimated at 30,000, based on the ratio of hybridization positive plaques to the total number of plaques screened, the average insert size, and the haploid genome of Zea mays, approximately 2.7 x 109 bp (K. Arumuganathan et al., Plant Mol. Bio. Rep. 9:208-218, 1991).

We have cloned and sequenced two new MPUs from maize and a recent report to GenBank shows a single MPU at approximately 2500 base pairs upstream from the Cat3 (catalase) gene (also of maize) (M.L. Abler and J.G. Scandalios, GenBank sequence entry MZECAT3GN). Interestingly, this is at about the same upstream location of the MPUs found in the zein gene pMS2, and both genes are highly regulated, being expressed only in germinating aleurone and developing endosperm tissues respectively. We have recently been informed of a cluster of four MPUs located about 3,000 bp upstream of the Knotted-1 gene of maize (J. Mathern and S. Hake, personal communication). Comparison of the first seven known perfect MPUs shows an almost perfect identity for the palindrome region (Figure 1), but not for their flanking regions, indicating that the MPU itself is the repetitive element. The MPU region of the Knotted-1 gene, however, has near perfect identity to the MPU region of pMS2.

Preliminary sequence data obtained from partial sequencing of one of the MPU-containing lambda clones reveals considerable homology to several potential reverse transcriptases. Figure 2 compares our sequence to reverse transcriptases from Zea mays (Z.m. rt) (D.F. Voytas, et al., PIR Release 41.0 accession number S27768), Drosophila (D.m. rtro) (F. Fourcade-Peronnet, et al., PIR Release 41.0 accession number S00954), and Volvox (W.c. rt) (A. Lindauer, et al., PIR Release 41.0 accession number S32437). Note that the amino acids conserved between the three reverse transcriptases are also conserved in our recently sequenced open reading frame. Further work is currently in progress to identify additional new genes adjacent to MPUs, and to indicate the function of this new DNA motif.

Figure 1. Comparison of the 7 known perfect MPUs. The palindromes are underlined. The center base of each palindrome is double underlined. The dashed lines between the palindromes represent perfect homology between corresponding MPUs.

Figure 2. Comparison of 4 coding regions from Zea mays, Drosophila, and Volvox. 17 of 89 amino acids (19%) are identical between the sequences.

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