Cold Spring Harbor Laboratory

Gene amplification at the P-wr allele

--Prasanna Athma and Thomas Peterson

P-wr is one of the few P alleles with differential expression in floral tissues: the P-specified pigments are present in cob glumes and absent in the pericarp. Preliminary results from Southern hybridizations indicate that the P-wr allele contains a 5-10 fold repetition, or amplification, of sequences present in P-rr. P-rr, which specifies red pericarp and cob, contains two 5.2 kb direct repeats separated by a 7 kb transcribed region (Lechelt et al., Mol. Gen. Genet. 219:225-234). The region amplified in P-wr represents most of the 7 kb transcribed region and one or both of the direct repeat sequences. Interestingly, a 700 bp fragment at the 3' end of the P-rr gene does not detect the P-wr amplified region; however, probes flanking this 700 bp fragment do hybridize to the amplified region. When restriction fragments outside the direct repeats were used as probes, the P-wr amplified region was not detected; instead similar banding patterns were observed in P-wr and P-rr alleles, indicating that some of the restriction sites are conserved in both alleles. Taken together, the Southern results suggest that the region amplified in P-wr represents approximately 12 to 21 kb of sequences present in P-rr. We do not know yet, either the size of the complete amplified unit or the arrangement of the repeats in the amplified region. Further experiments to determine the structure of the P-wr amplified region are in progress.

Since gene amplifications in other organisms are associated with developmentally regulated gene expression, we looked for differences in the P-wr amplification in different tissues of the maize plant. DNA was prepared from tissues in which P is expressed (silks, husks, cob glumes) or not expressed (leaves), digested with restriction enzymes, and analyzed by Southern hybridizations. No obvious differences in the intensities or sizes of the P hybridizing bands in the different tissues were found. Therefore, the P-wr amplified structure is not detectably altered during development. The P-wr transcriptional pattern is under investigation.

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