Isolation of a candidate clone of the maize P locus

The P gene controls pigmentation of the pericarp and glumes of the cob and tassel. Many variants in P expression are known, including types displaying altered tissue specificity, and variegated types. Schwartz has presented a model of P expression (D. Schwartz, Proc. Natl. Acad. Sci. 79:5991-5992, 1982) involving a hypothetical regulatory mechanism termed presetting by McClintock (B. McClintock, Carnegie Inst. Washington Yearb. 63:592-601, 1964).

The P-VV allele, which conditions variegated pericarp, has been shown by Brink and others to result from the effects of the transposable element Mp (functionally equivalent to Ac) situated at the P locus. Since the Ac element has been cloned previously (Fedoroff et al., Cell 35:235-242, 1983), Ac-specific sequences can be used to isolate the P-VV allele.

DNA prepared from maize seedlings carrying a P-VV allele was digested with restriction endonuclease SalI. The SalI fragments were ligated into SalI-cut phage vector EMBL3. The resulting library was screened with a probe prepared from an internal fragment of the Ac element. A hybridizing clone was detected which carried an 8 kilobase pair SalI fragment containing a full-length Ac element. Sequences flanking the Ac element were used as probes for Southern blot analysis of DNA obtained from stocks carrying P-VV (variegated pericarp) and a P-RR (red pericarp) allele derived as a revertant of the unstable P-VV allele. When these DNAs were restricted with SalI and probed with the presumptive P-specific probe, the P-VV DNA contained an 8 kilobase pair-hybridizing band, while the P-RR DNA contained a 3.5 kb band. This result is consistent with the presence of the 4.5 kb Ac element in the P-VV allele, and loss of the Ac element upon reversion to a P-RR allele.

The DNA flanking the cloned Ac element was sequenced. Surprisingly, the Ac element was not associated with a short (8 bp) direct repeat found flanking other Ac elements. The generation of short direct repeats upon insertion is a feature of nearly all transposable elements studied to date, and the significance of their absence in this particular case is unknown.

Starlinger and coworkers are also studying the P locus (see report in this issue). They report the isolation of an 8 kb SalI fragment derived from P-VV. The independently isolated fragments are apparently identical, and sequence comparisons indicate that in both cases the Ac element is situated at the same nucleotide position, lacking an 8 bp repeat.

The pedigree relationship of the stocks used by each group is unclear, but we assume that these P-VV alleles are identical. (This material is based upon work supported by the National Science Foundation under a grant awarded in 1984).

Thomas Peterson and Drew Schwartz

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