We studied the transcription of the Adh1-2F11::Ds2 allele. In this allele a Ds2 element (described in MNL 1986 by A. Merckelbach and P, Starlinger) is inserted in the fourth exon of Adh1.
In Northern blot hybridizations with an Adh1-specific probe two bands light up:
1. A 3 kb transcript that has the combined length of wildtype Adh1 RNA plus the length of the insertion.
2. A transcript of about 1.6 kb (described in MNL 1983 by S. Hake and M. Freeling).
Only the 3 kb band is detected with a Ds2-specific probe. The 1.6 kb RNA cannot be a transcript of an Adh1 allele created by the excision of the Ds2 element as it was also seen in Ac-free maize lines.
By Southern blot hybridizations it was confirmed that the Ds2 element still resides at the Adh1-locus. For further analysis of the 1.6 kb RNA we prepared a cDNA library and screened for clones that hybridize to Adh1 but not to Ds2 probes. Recombinant phages were partially sequenced around the insertion site of the Ds2 element in Adh1.
The cDNA clones sequenced are derived from RNA molecules that have lost 132 bp of the 5' part of exon 4 including the whole Ds2 sequence. This mRNA molecule must be the result of a splicing of the normal 5' donor splice site at the beginning of intron 3 to a cryptic 3' acceptor splice site in exon 4. The acceptor splice site used is not visibly inferior to the one terminating intron 3.
Is this cryptic splice site also used in the wildtype or is it activated only in the 2F11 allele by the Ds2-element? To test this we did Northern blot experiments with a synthetic oligonucleotide that spans the abnormal splice junction and thus can hybridize only to the aberrantly spliced 1.6 kb RNA molecules. This oligonucleotide probe detected a 1.6 kb mRNA in Adh1-2F11, but not in wildtype material.
Some other abnormal transcripts of the Adh1-2F11 allele were detected. One group of cDNA clones was derived from RNA molecules that were terminated and polyadenylated at two different sites in intron 3. In addition one cDNA clone that had lost the Ds2 element and part of exon 4 by aberrant splicing was terminated in intron 6.
This analysis shows that the insertion of a Ds2 element can drastically alter transcription termination and the splicing pattern at a distance to the insertion site.
Rudiger Simon and Peter Starlinger
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