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Molecular biology. Transformation: introduction of DNA Selectable marker Spheroplasts, Li 2+ salts, electroporation Yeast plasmids are shuttle plasmids, amplification in E. coli, ori, b -lactamase Transformation with oligonucleotides, -> selection

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Molecular biology l.jpg

Molecular biology

Transformation: introduction of DNA

Selectable marker

Spheroplasts, Li2+ salts, electroporation

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

ori, b-lactamase

Transformation with oligonucleotides, -> selection

Transformation of mitochondria by particle bombardment


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Important genes

  • URA3, LYS2, can be negative selected against (FOA, a-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 ade3ade2 is white

    • Colony sectoring screen

    • Syntehtic lethal secreen

  • GAL promoter for conditional expression

  • lacZ, GFP fusions

  • Epitope tags


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Homologous recombination gene disruption

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.

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.

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.





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Allele rescuegap repair


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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



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Nomenclatureof geneticinteractions


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Reverse genetics

  • Gene -> phenotype -> function (annotation)


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Yeast specific Methods

  • Two hybrid

  • Yeast Artifical Chromosomes (YACs) (50- 500kb)

  • Expression of heterologous proteins

    • No endotoxins

    • Posttranslational modifications (acetylation, myristoylation,..)

    • secretion






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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


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Gene-omics

Microarrays (DNA, oligo)

comparative, yeast (sensus stricto),...

Proteomics, MS, chips (120 kinases)

Metabolomics, flux of metabolites


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microarrays

  • microarray tutorial



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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