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Molecular biology

Molecular biology

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Molecular biology

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  1. 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

  2. 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

  3. 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.

  4. Two-step gene replacement

  5. 4 types of yeast vectors

  6. Plasmid shuffling

  7. Allele rescuegap repair

  8. 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

  9. Synthetic enhancement and synthetic lethality

  10. Nomenclatureof geneticinteractions

  11. Reverse genetics • Gene -> phenotype -> function (annotation)

  12. Yeast specific Methods • Two hybrid • Yeast Artifical Chromosomes (YACs) (50- 500kb) • Expression of heterologous proteins • No endotoxins • Posttranslational modifications (acetylation, myristoylation,..) • secretion

  13. YACs

  14. Recombination cloning of YACs

  15. The two-hybrid system

  16. Protein interaction map

  17. 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

  18. Gene-omics Microarrays (DNA, oligo) comparative, yeast (sensus stricto),... Proteomics, MS, chips (120 kinases) Metabolomics, flux of metabolites

  19. microarrays • microarray tutorial

  20. clustering

  21. 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