Molecular analysis of the P locus

We have continued our work on the structure of the P gene. We have isolated a 14 kb EcoRI clone extending from the EcoRI site in Ac to an EcoRI site located in the direction of the 5'-end of the Ac transcript. Near the end of this clone, a 1.2 kb Sal1 fragment is located that is seen in Southern hybridizations of genomic DNA of P-vv, P-rr and a revertant from P-vv to P-rr.

From a P-rr library, two MboI clones of 13.5 and 14 kb length were isolated that overlap with the DNA sequence located beyond the 3'-end of Ac on the original 8 kb Sal1 fragment. This clone does not contain any further sequences hybridizing to the three Sal1 fragments of 1.2 kb, 3.0 kb and 3.4 kb length hybridizing to the DNA flanking the Ac insertion in P-ww.

A cDNA was cloned from a library constructed from pericarp RNA of P-rr kernels 24 days after pollination. It is 503 bp in length and is supposed to represent the 3'-terminus of (a) larger transcript(s). It is nearly 100% homologous to a genomic region located 2 kb 3' of the Ac insertion site. The direction of transcription is determined by this cDNA clone.

Using the cDNA sequence in a single strand specific M13 vector as a probe in the RNA analysis of different P alleles, two large P-rr and P-revertant specific transcripts of 7 kb and 12 kb in size were detected. These are not found in P-vv and P-ww derived RNA. The two transcripts were also detected with a single strand specific probe derived from a 400 bp fragment located directly 5' of the Ac insertion site. The same probe also detects an 8 kb and very weak >12 kb transcript in the RNA of P-vv. These transcripts were also detected by a single strand specific probe derived from Ac.

They represent chimeric molecules consisting of Ac- and P-derived sequences The direction of transcription of the P gene and Ac is identical in P-vv. The data are in agreement with the hypothesis that the gene is terminated at the location of the polyadenylation of the Ac transcript (Kunze et al., EMBO J. 6:1555, 1987). In support of this hypothesis, the transcripts from P-vv do not hybridize to the cDNA clone.

It is noteworthy that this result excludes the 1.2 kb Sal1 band located on the other side of Ac from these transcripts and that no other transcripts hybridizing to this DNA have yet been detected.

Christa Lechelt and Peter Starlinger


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