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A yeast-based model system for cloning secreted and membrane proteins
Marcelo J Surpili1, Bernd Müller-Röber, Lothar Willmitzer
1Institut für Genbiologische Forschung Berlin GmbH, Berlin, Germany. msurpili@lnls.br
Anais Da Academia Brasileira De Ciencias
|February 4, 2003
Summary
Researchers developed a novel signal sequence trap system using yeast acid phosphatase. This method successfully identified two unknown plant sequences with specific tissue expression patterns, aiding in gene discovery.
Area of Science:
- Molecular Biology
- Plant Science
- Genetics
Background:
- Protein targeting to organelles and secretion relies on signal sequences at the 5'-end of genes.
- A signal sequence trap system can be a powerful tool for identifying novel genes based on their regulatory elements.
Purpose of the Study:
- To develop and validate a signal sequence trap system for identifying novel plant genes.
- To screen a potato guard-cell cDNA library for sequences encoding functional signal peptides.
Main Methods:
- A truncated yeast acid phosphatase (pho5) gene lacking its signal sequence was used as a reporter.
- cDNA fragments from a potato guard-cell library were cloned in-frame to the reporter gene.
- Acid phosphatase activity in yeast colonies was used to assay for functional signal sequences.
Main Results:
- The system successfully identified two unknown potato cDNA sequences (YE139 and YE290) with distinct tissue-specific expression.
- Sequence YE139 showed higher expression in green buds and stem cells.
- Sequence YE290 exhibited higher expression in reproductive tissues (androecium, gynoecium) and roots.
Conclusions:
- The developed signal sequence trap is effective for identifying novel genes with specific expression patterns in plants.
- The retrieved sequences YE139 and YE290 represent potentially important genes in potato development and reproduction.
- The study highlights the utility of reporter gene systems for functional genomics in plants.