Cloning of DNA from the P locus

A wildtype of the P gene, P-rr, is necessary for the pigmentation of the pericarp and the cob. In some alleles, either pigmentation of the pericarp or of the cob is lost independently (P-wr and P-rw). In the P-vv allele, however, the insertion of Mp (equal to transposable element Ac), leads to the loss of both pericarp and cob pigmentation. Excision of Mp from the locus restores pigmentation of both tissues simultaneously. Thus, in this allele P behaves as a single gene (Brink and coworkers). As a prerequisite for studies of this interesting gene, we have cloned DNA of this locus from both P-vv and P-rr.

For the cloning of P-vv, we used a central segment of Ac as a probe. We identified an 8 kb SalI fragment absent from P-ww. This fragment was cloned and shown to contain a 4.5 kb insertion identical to Ac by restriction analysis and partial DNA sequence.

A 2.6 kb BgIII-SalI fragment flanking the Ac element was used as a probe for the identification of P sequences in the P-rr allele. P-rr contains a 3.5 kb SalI band absent from P-vv. This band is also absent from the null mutation P-ww, which may thus be a deletion. The 3.5 kb band is cleaved by BgIII yielding a 2.6 kb fragment. This is expected from the DNA prior to the insertion of Ac. In addition to the 3.5 kb band, several other bands also hybridize to the flanking probe. The relation of these to the P gene is not known at present.

Partial sequencing of the termini of Ac inserted in the P sequences shows two interesting features:

1. The outermost nucleotides of Ac are not complementary to each other and resemble in this respect the two cloned copies of Ac from wx-m9 (R. Pohlman et al., Cell 37:635, 1984) and from wx-m7 (M. Muller-Neumann et al., MGG 198:19, 1984). As the P-vv allele was isolated by Emerson as early as 1917, while the two Ac elements in the waxy gene derive from an Ac activated from a silent state by McClintock, this similarity either shows that this difference of Ac to most Ds elements has a functional meaning or else that all the Ac elements cloned so far are evolutionarily closely related.

2. The absence of an 8 bp flanking duplication that is found accompanying all other Ac or Ds insertions possibly indicates the formation of an adjacent deletion after the insertion. The reversion of P-vv to P-rr must then tolerate this deletion event, possibly because the insertion has occurred in an intron. Peterson and Schwartz describe a clone of P-vv DNA identical to our clone (see report in this issue).

From P-rr DNA, lambda clones hybridizing to the above mentioned flanking probes have been isolated and are presently under investigation. The cloning of the P locus has also been performed by J. Chen and S. Dellaporta, Cold Spring Harbor (personal communication).

Christa Lechelt, Alan Laird and Peter Starlinger
 
 


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