Molecular analysis of the P locus

--Christa Lechelt and Thomas Peterson

The maize P locus controls pigmentation of certain floral tissues, including the pericarp and glumes of the cob. Interestingly, P can be expressed independently in pericarp and cob; thus, the P-RR, P-RW, P-WR, and P-WW alleles specify red pericarp/red cob, red pericarp/white cob, white pericarp/red cob, and white pericarp/white cob, respectively. The P-VV allele, which specifies variegated pericarp and cob, comprises the transposable element Ac inserted in a P-RR gene.

Using the Ac element as a transposon tag we have isolated 27kb of genomic DNA from the P locus. With an overlapping genomic BamHI clone of 10kb (kindly provided by Jychian Chen and Stephen Dellaporta) the entire cloned region comprises 34kb. The cloned DNA contains two 5.8kb homologous regions, in direct orientation, separated by 6.6kb. The Ac element in the original P-VV allele is inserted in the 6.6kb of DNA between the 5.8kb direct repeats.

We knew from previous experiments that the Ac insertion in P-VV is correlated with a change in transcriptional pattern around the Ac insertion site (MNL 62:47). For a complete transcriptional analysis of the cloned P-locus DNA, restriction fragment probes spanning the 34kb region were hybridized to Northern blots of RNA from plants carrying the mutant P-VV and functional P-RR alleles. The results allowed a coarse determination of transcribed regions that are most probably specific for the P gene.

We found that probes from a region of 7.3kb around the Ac insertion site detect five transcripts of 7kb, 6.5kb, 2kb, 1.4kb and 1kb in RNA of P-RR and P-RR revertants derived from P-VV. The multiple RNA molecules may be formed by differential splicing. None of these transcripts is found in RNA from the P-VV allele. Instead, a transcript of 9.5kb in size is detected in P-VV RNA probes located 5' of Ac and by Ac-specific probes. The 9.5kb RNA is a chimeric transcript containing P- and Ac-specific sequences that most likely terminates within the Ac element, since it is not detected by probes 3' of Ac. Hybridization with single strand specific M13 probes demonstrated that the direction of transcription of the P gene is identical to the Ac gene in the cloned P-VV allele (MNL 62:47). Thus, the transcriptional start site(s) of the P gene is located 5' of the Ac element in the P-VV allele used for these studies. The transcript sizes reported previously (MNL 62:47) are overestimates, and the sizes given here are derived by side by side comparison with labelled RNA ladder and HindIII-cleaved lambda DNA.

Probes made from DNA fragments outside the 7.3kb region around the Ac insertion site do not detect differences in RNA from the P-VV and P-RR RNA. These transcripts do not seem to be specific for the P gene since they do not correlate with the phenotype, but we cannot exclude the possibility that they are somehow involved in P gene expression.

Although the 7.3kb region around the Ac insertion site is able to code for the largest transcript of 7kb, we do not yet know whether the promotor of the P gene is also located in this region, or whether RNA synthesis starts further 5'. At present it is also unknown which of the five transcripts are important for the expression of the P gene, or whether all of them may be.


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