Complementation of the salt sensitive S. cerevisiae mutant cch1 with an A. thaliana cDNA library and analysis of isolated genes

The yeast leucine heterotrophic wild-type strain DBY689 had been knocked out in the gene CCH1 obtaining a salt stress sensitive phenotype. For screening an Arabidopsis cDNA library selection conditions were established. On the optimized media it was possible to distinguish between the growth rates of the wild type, transformed with a control plasmid, and the knock-out mutant, transformed with a control plasmid, which exhibits much smaller colonies. Clones of cch1 transformed with the cDNA library showing a similar growth rate like the wild type, were isolated and tested by re-transformation upon their ability for complementation. <br>A total of 16 isolated clones were confirmed twice to complement the knock-out cch1 and could be categorized by their quality of complementation. Furthermore they were tested for surviving on media with toxic levels of LiCl. The transformants were classified into two complementation groups, one of which (class I) showed similar growth to the wild type and was studied extensively. The six transformants belonging to this class I were tested upon their growth ability in low calcium medium and furthermore for sensitivity to the yeast alpha-factor. The transcripts of the isolated cDNAs were analyzed by Northern blot detection, and localization studies with GFP fusion protein were performed. In addition fragments from the promoter regions of several cDNA clones were fused to the luciferase and the beta-glucuronidase gene and integrated into the Arabidopsis genome. The obtained results indicate that the isolated genes are involved in a more general response to abiotic stresses since inductions after salt, cold and ABA treatments could be detected.

Author dc:creator

• Markus, Ralf

Contributors dc:contributor

• Neuhaus, Gunther

• Hefe
• Salz
• yeast
• salt

Repository record source_url

https://freidok.uni-freiburg.de/data/219

OAI identifier oai:identifier

oai:freidok.uni-freiburg.de:219