Transcript PowerPoint-Präsentation

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
Important genes
• URA3, LYS2, can be negative selected against (FOA, aaminoadipic 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
Homologous recombination gene
disruption
(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.
Two-step gene replacement
4 types of yeast vectors
Plasmid shuffling
Allele rescue
gap repair
Interactions of genes
• Physical interactions, two-hybrid, co-Ips, co-purification...
• Genetic interactions, synthetic lethal, supression, dominantnegative
• 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
Nomenclature
of 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
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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
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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