CELL CYCLE Flashcards
a) What are cell fusion experiments?
b) What do cell fusion experiments show? Specific experiments…
a) fusing together cells at different cell cycle stages
b) fusion with a mitotic cell ALWAYS triggers premature chromosome condensation
- interphase + mitotic cell -> premature mitosis
- G1 and S1 phase -> driven into S phase
BUT…
S phase + G2, no new DNA synthesis
Why do we need model systems for cell cycles?
- cell division is very complicated
- genetically trackable
- study impacts of conditional mutants
What are conditional mutants? Key features
- recessive and loss of function
- have permissive and restrictive conditions
Why are conditional mutants useful?
- can use to clone the WT gene
- look for simple genetic interactions
Latin name for fission yeast
Schizosaccharomyces pombe
Latin name for budding yeast
Saccharomyces cerevisiae
Distinguish between fission and budding yeast in terms of genetic screening
FISSION - good for G2, division occurs down middle and forms a septa
BUDDING - transition from S phase and mitosis is less distinct, meaning these aren’t fantastic for observing G2
What are cdc mutants?
cell division cycle
How were cdc mutants used to demonstrate temperature sensitivity in budding yeast?
- induced mutagenesis in WT budding yeast found a large number of mutants that arrest at specific points of the cell cycle at a certain temperature (36 degrees)
When do budding yeast cdc mutants arrest?
end of G1
How do you clone a WT gene from a budding yeast cell that is mutant?
- transform mutant with entirety of cDNA/ genome library (insert sequences into plasmids and into mutant cells)
- identify colonies that can grow at 37 degrees
- purify plasmid and sequence insert
Example of shuttle vector that is useful in cloning genomic libraries?
Yeast CEN
a) What is yeast gene knockout?
b) What is it used for?
c) How does it operate?
d) Host receives the ___ but no longer has the __ of interest.
a) also known as positive drug selection, used to manipulate cloned genes
b) to observe null phenoytpe
c) - the yeast plasmid is linearised and a drug resistance gene is inserted that is flanked by identical restriction sites that flank the gene of interest
- two plasmids mixed and homologous recombination is used to swap out the gene of interest for the drug resistance gene
d) marker
gene
What is an epitope?
the part of an antigen molecule to which an antibody attaches itself.
PCR- mediated epitope tagging is all about what?
_____ then bind to this entity and reveal it.
protein product
antibodies
