Lecture 4 - Regulation of replication Flashcards
Describe why budding yeast is used as a model organism
Unicellular eukaryote (life cycle = cell cycle)
Easy to grow with visual markers of cell cycle
Genetic analysis and synchrony possible
Different points in the cell cycle are visible externally - buds are different sizes depending on where the cell is in the cell cycle - can see what stage a cell/population of cells has been arrested
Easy to grow - economically a useful research organism as they can be grown in large quantities for analysis
Can intervene in the yeast cell cycle and synchronise their growth: they generate pheromones (mating factors) which can synchronise other yeast cells. Arrest at START point - known as restriction point R in mammalian cells - main control point that is deregulated in cancer cells.
Describe how you screen for temperature sensitive mutants
Created populations of yeast cells that were capable of growing under normal conditions but not in restrictive temperature - amino acid changes that affect the thermodynamic properties of protein coded for by that gene - temperature-sensitive/conditional mutants. Mutants were classified according to where in the cell cycle they arrest when put in the restrictive temperature. Frequently the cdc mutants arrested at a point of the cell cycle that preceded the point of action of the gene product - came up with notion of checkpoint.
a cell division cycle (cdc) mutant arrests before the phase in which its gene product is required
non- cell cycle division mutant arrests immediately irrespective of cell cycle stage
What is plasmid rescue?
Make cDNA library from transcriptome of wild-type population of yeast - whole population of plasmids that encode for all the transcipts made by yeast. Transformed this cDNA library into mutant yeast population and looked for ones that could then grow at restrictive temperature. Only the cells in receipt of a gene to complement their defect will grow at restrictive temperature. Went on to re-isolate cDNA that conferred that normal growth and therefore the gene that underpinned the defect in the first place.
