To investigate if the maize element Activator (Ac) can transpose in fission yeast (Schizosaccharomyces pombe), we have constructed shuttle vectors containing a potentially autonomous Ac derivative inserted between a promoter and a selectable marker gene.
Two major modifications were introduced into Ac. In order to ensure expression of the Ac transposase gene in S. pombe, we replaced the Ac promoter and leader sequence with the fission yeast nmt1-promoter. This promoter can be repressed by growing the cells in thiamine-containing medium, which could be of advantage in case that expression of the Ac transposase at high levels turns out to be harmful for the cells. In addition, to circumvent potential splicing problems in fission yeast, the genomic transposase coding region of Ac was replaced by the corresponding cDNA sequence (ORFa).
This modified Ac was inserted between the cauliflower mosaic virus 35S promoter and the NPTII gene, which are known to be functional in fission yeast. The final constructs contain the ura4 gene as selectable marker and were transformed into the S. pombe strain h- ura4-D18. Excision of the modified Ac should be detectable by G418-resistance of the cells.
By Northern analysis of the transformed cells, three ORFa-homologous transcript bands were detected. The strongest signal appeared as a rather broad band about 2.8 kb in length, which is the expected size for a polyadenylated, full length ORFa transcript. In addition, two distinct but less intense bands, 1.9 kb and 3.4 kb in length, hybridized with the ORFa probe. These RNAs are presumably derived by transcriptional read-through of the Ac polyadenylation signal as they hybridize with Ac sequences 3´ to this site. The transcription initiation sites of the transcripts have not been determined.
On Western blots several protein bands are recognized by an anti-transposase-serum in total protein extracts of the transformed fission yeast cells. The largest, very weak band has the same electrophoretic mobility as the wild-type transposase. The major fraction of ORFa-derived proteins apparently consists of aberrantly processed transposase molecules.
After plating transformed yeast cells on G418-containing medium we have recovered several G418-resistant colonies. However, a simple excision of the modified Ac had not occurred in any of the reisolated plasmids, but rather recombinational deletions between short, directly repeated sequences were found.
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