Molecular biology Transformation: introduction of DNA – Selectable marker – Spheroplasts, Li 2+ salts, electroporation Yeast plasmids are shuttle plasmids,

Molecular biology

• Transformation: introduction of DNA– Selectable marker

– Spheroplasts, Li2+ salts, electroporation

• Yeast plasmids are shuttle plasmids, amplification in E. coli,

–ori, -lactamase

• Transformation with oligonucleotides, -> selection

• Transformation of mitochondria by particle bombardment

Important genes

• URA3, LYS2, can be negative selected against (FOA, -aminoadipic acid)– Host alleles, ura3-52 -> ura3∆0

• Dominant drug selectable markers, i.e. kanMX• ADE1, ADE2, ade1 and ade2 mutants produce a red pigment

– But ade3 ade2 is white– Colony sectoring screen– Syntehtic lethal secreen

• GAL promoter for conditional expression• lacZ, GFP fusions• Epitope tags

(A) The YFG1 +gene is disrupted by transforming the strain with a linear fragment containing a URA3 selectable marker flanked by homologous sequences. The chromosomal segment is replaced by this URA3 containing fragment after integration by homologous recombination.

(B) The URA3 marker introduced in the YFG1 locus, can be excised if URA3 is also flanked by direct repeats of DNA, preferably not originating from yeast. Homologous recombinants lack the URA3 marker and retain a single copy of the repeated DNA.

(C) Single-step gene replacement of mutant alleles, such as yfg1-1 , can be carried out by first replacing the YFG1 gene by URA3 , transforming the strain with linear fragment encompassing the yfg1-1 mutation, and selecting transformants in which URA3 is replaced by yfg1-1.

Homologous recombination gene disruption

Two-step gene replacement

4 types of yeast vectors

Plasmid shuffling

Allele rescuegap repair

Interactions of genes

• Physical interactions, two-hybrid, co-Ips, co-purification...• Genetic interactions, synthetic lethal, supression, dominant-

negative

• Intragenic complementation• Non-allelic non-complementation• Suppressors

– Informational, ie tRNAs (are allele but not gene specific)– Metabolic, gene specific, bypass suppressors

• Synthetic enhancement, epistasis

Synthetic enhancement and synthetic lethality

Nomenclatureof genetic

interactions

Reverse genetics

• Gene -> phenotype -> function (annotation)

Yeast specific Methods

• Two hybrid• Yeast Artifical Chromosomes (YACs) (50- 500kb)• Expression of heterologous proteins

– No endotoxins– Posttranslational modifications (acetylation,

myristoylation,..)– secretion

YACs

Recombination cloning of YACs

The two-hybrid system

Protein interaction map

Cell biology

Mutant collections Cell division cycle mutants, cdc Pre-mRNA splicing mutants, prp Secretory mutants, sec Vacuolar protein sorting mutants, vps Sterile mutants, ste Endocytotic mutants, end

Methods: GFP Pulse-chase Genetic interactions

Gene-omics

• Microarrays (DNA, oligo)

• comparative, yeast (sensus stricto),...

• Proteomics, MS, chips (120 kinases)

• Metabolomics, flux of metabolites

microarrays

• microarray tutorial

clustering

SAGE

• ACO T56

• Cell 1997, 243

• Transcripts 0.3 - 200 / cell

• Only 18% of genes have > 100 transcripts (energy metab. ribosome)

• No transcript clustering in genome