Genomic Southern analysis indicates that, similar to P1-rr, P1-rw contains a single copy gene flanked by long direct repeats. RT-PCR results showed that the P1-rw 5’ UTR and 5’ coding sequences are identical to P1-rr and P1-wr. However, the P1-rw 3’ coding sequences and 3’ UTR are identical to that of the p2 gene, a tightly linked, paralogous gene that regulates the synthesis of 3-deoxyflavones, including maysin, in maize silk (Zhang et al, The Plant Cell 12:1-12, 2000; Zhang et al., Plant Molecular Biology, in press.) To reconcile these results, we screened a genomic lambda library with a probe specific for P1-rr (Fragment 15), and a second probe which hybridizes with both p1 and p2 (Fragment 8B). Two classes of lambda clones were isolated from the library: the first class resembles p2; these clones hybridize only with probe 8B, and they lack an 80 bp indel in the 5’ UTR sequence. The second class resembles p1; these clones hybridize with both probe 15 and 8B, and they contain the 80bp indel in the 5’ UTR sequence. Two overlapping p1-like lambda clones were sequenced and found to match the sequences of P1-rw transcripts detected by RT-PCR. Interestingly, although P1-rw is structurally similar to that of P1-rr (both contain a coding sequence flanked by two long direct repeats), P1-rw differs from P1-rr in two aspects: First, the P1-rw coding sequence is chimeric, and consists of a p1-like 5’ UTR, followed by p2-like exons and introns, followed by a truncated P1-wr-like exon 3 (Figure 1). This chimeric structure appears to have been generated by recombination between the p1 and p2 genes. Second, P1-rw has a major polymorphism in the 1.2 kb distal enhancer region which is located 5 kbp upstream of the p1 transcription start site; specifically, a 339 bp sequence located between the PstI and SacI sites in the P1-rr distal enhancer was replaced by a distinct 71bp fragment in P1-rw.
Our current hypothesis is that polymorphisms in the coding sequences and/or the distal enhancer region are responsible for the distinct gene expression pattern of P1-rw. Nuclear run-on assays are underway to test whether P1-rw expression is regulated at the transcriptional or post-transcriptional level, or both.
Figure 1. Structure of the P1-rw allele. P1-rw appears to be a chimeric gene generated by recombination between p1 and p2 sequences: the p2-like coding sequences are flanked by p1-like long direct repeats (open boxes). Exons and introns are indicated by black boxes and bent lines, respectively; the bent arrow indicates the transcription start site experimentally determined for the P1-rr allele (Grotewold et al., Proc. Natl. Acad. Sci. 88:4587-4591, 1991). The hatched box indicates a truncated P1-wr-like exon3. Sequence polymorphisms in the distal enhancer region are shown as dotted black boxes (339bp in P1-rr, 71 bp in P1-rw).
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