Cloning and characterization of P-ww*-12:27-3 allele
--Jianbo Zhang and Thomas Peterson

In last year's Maize Newsletter (68:10), we reported a new P-ww allele of the P gene with interesting characteristics. The P-ww*-12:27-3 allele suppresses the orange pericarp pigmentation specified by P-ovov-1114, and has a very strong negative Ac dosage effect. To determine the structure of the P-ww*-12:27-3 allele, a genomic library was constructed in lambda FIX II. Ten clones were isolated which hybridized to probes from both Ac (the internal 1.6 kb HindIII fragment) and the P gene (JZ001, a PCR product within P-rr fragment 10). Five clones (Type I) also hybridized with P-rr fragment 8B, while the other five clones (Type II) did not. Southern blot and PCR analysis indicated that Type I clones contain an inverted duplication of part of the P-rr gene, which is consistent with genomic Southern blots, and could suggest that P-ww*-12:27-3 inhibits P-ovov-1114 expression through an anti-sense RNA mechanism. The duplication in P-ww*-12:27-3 is at least 10 kbp in length, beginning in P-rr fragment 10 and extending beyond the EcoRI site located 6 kbp 5' of the P-rr transcription start site (see Figure 1). Type II clones contain most or all of an apparently normal Ac element, and an unknown DNA fragment which is probably the extension of the duplication present in Type I clones. Northern blot analysis showed that the pattern of Ac transcription in P-ww*-12:27-3 is different from that in P-vv and P-ovov, but the mechanism of the high negative Ac dosage effect of P-ww*-12:27-3 remains to be determined.

Figure 1. Restriction map of the P-ww*-12:27-3 allele. The central map indicates the structure of the P-rr allele, with 5.2 kb repeats (hatched boxes) in direct orientation flanking the P-rr transcribed region. The P-ww*-12:27-3 allele contains two insertions within P-rr fragment 10: an Ac element (upper triangle), and a large complex insertion (lower triangle) containing both Ac-homologous sequences (black boxes) and P-homologous sequences in inverted orientation. The different dashed lines represent different lambda clones: clones 16, 21, and 5 are Type I clones, clones 22 and 17 are Type II clones. Restriction enzyme sites are indicated as follows: B = BamHI, Bl = BglII, E = EcoRI, H = HindIII, K = KpnI, P = PstI, S = SalI, Sa = SacI, X = XhoI. Not all sites for PstI and SacI are shown.

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