Immunolocalisation of two tropinone reductases in potato (Solanum tuberosum L.) root, stolon, and tuber sprouts.

Tropinone reductases (TRs) are essential enzymes in the tropane alkaloid biosynthesis, providing either tropine for hyoscyamine and scopolamine formation or providing pseudotropine for calystegines. Two cDNAs coding for TRs were isolated from potato (Solanum tuberosum L.) tuber sprouts and expressed in E. coli. One reductase formed pseudotropine, the other formed tropine and showed kinetic properties typical for tropine-forming tropinone reductases (TRI) involved in hyoscyamine formation. Hyoscyamine and tropine are not found in S. tuberosum plants. Potatoes contain calystegines as the only products of the tropane alkaloid pathway. Polyclonal antibodies raised against both enzymes were purified to exclude cross reactions and were used for Western-blot analysis and immunolocalisation. The TRI (EC 1.1.1.206) was detected in protein extracts of tuber tissues, but mostly in levels too low to be localised in individual cells. The function of this enzyme in potato that does not form hyoscyamine is not clear. The pseudotropine-forming tropinone reductase (EC 1.1.1.236) was detected in potato roots, stolons, and tuber sprouts. Cortex cells of root and stolon contained the protein; additional strong immuno-labelling was located in phloem parenchyma. In tuber spouts, however, the protein was detected in companion cells.

Rad51p and Rad54p, but not Rad52p, elevate gene repair in Saccharomyces cerevisiae directed by modified single-stranded oligonucleotide vectors.

Synthetic single-stranded DNA vectors have been used to correct point and frameshift mutations in episomal or chromosomal targets in the yeast Saccharomyces cerevisiae. Certain parameters, such as the length of the vector and the genetic background of the organism, have a significant impact on the process of targeted gene repair, and point mutations are corrected at a higher frequency than frameshift mutations. Genetic analyses reveal that expression levels of the recombination/repair genes RAD51, RAD52 and RAD54 can affect the frequency of gene repair. Overexpression of RAD51 enhances the frequency 4-fold for correction of an episomal target and 5-fold for correction of a chromosomal target; overexpression of RAD54 is also effective in stimulating gene repair, to the same extent as RAD51 in the chromosomal target. In sharp contrast, RAD52 gene expression serves to reduce gene repair activity in rescue experiments and in experiments where RAD52 is overexpressed in a wild-type strain. This may suggest an antagonist role for Rad52p. Consistent with this notion, the highest level of targeted repair occurs when the RAD51 gene is overexpressed in a strain of yeast deficient in RAD52 gene function.