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Random insertional mutagenesis into yeast gDNA library.

Key mTn features:

• Met^- lacZ - creates fusion protein that can be detected with Xgal (no IPTG)
• URA3 - selection in yeast for presence of mTn
• tet - selection in E. coli for plasmid
• two lox sites - allows for Cre-mediated deletion of insertion
• 3xHA - three copies of the haemagglutinin epitope tag to which a monoclonal antibody binds, used to follow your favorite protein after Cre-lox excision.

What's Available?

• 11,232 stains created
• 6,358 strains verified by sequence
• 5,442 in or very near annotated ORFs
• 1,917 ORFs affected
• 1,826 strains have high ß-gal expression as fusion proteins
• 328 non-annotated ORFs (NORFs) discovered (due to small size or overlapping larger ORFs)

Different Levels of Analysis

1) Macroarrays

• Wild-type Expression Patterns "enhancer trap-like" (ß-gal)
• Specific Growth Conditions:

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2) Microarrays

• normal RNA-based method

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3) Proteomics

• epitope tag protein detection

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4) "Phenomics"

• Dissection of individual genes since more than one insertion per ORF
  (e.g. IMP2' transcription factor with 11 different insertion sites)
• Growth of individual stains on selective media

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5) Clustering of Phenomics

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Significant Conclusions for GCAT:

• Discover NORFs.
• Discover function of unknown ORFs.
• Discover unsuspected functions for known ORFs.
• Discover drug targets (direct and indirect).
• 11,232 strains available for further study.
• NO RNA ISOLATION NECESSARY!
• Little correlation between gene expression microarray data and selection data.
• Many strains and an infinite number of environmental conditions.

PLUS

• Domain mapping with multiple insertions.
• ß-gal fusion provides gene activation method.
• ade2 oxphos detection.
• Macroarray phenomics (96 well or on solid media).
• Proteomics with HA epitope tagging.
• Web-based TRIPLES database for 7800+ mutant strains and online ordering form for strains
  (Transposon-Insertion Phenotypes, Localization and Expression in Saccharomyces)

Caveats

• Not every ORF hit due to "random" mTn insertion.