Apoptosis Flashcards
(25 cards)
apoptosis (3)
- programmed/active cell death
- requires energy, RNA and protein synthesis
- cleared by phagocytosis; no inflammation or tissue damage
necrosis (3)
- passive cell death
- cells swell, membrane breaks down, contents leak, nucleus disintegrates
- inflammatory (tissue damage)
apoptosis functions (2)
- multicellular development: sculpting tissues, deletion of structures, regulation of neuron number
- body’s defence against cancer
is apoptosis evolutionarily conserved (2)
yes:
- occurs in all multicellular animals and plants
- stages and genes are conserved from worms and flies to mice and humans
what phenotype is expected if engulfment is blocked
- accumulation of cell corpses
characteristics of mutations affecting engulfment (3)
- cell death occurs at normal time, but cell corpses persist long after cells die
- DNA is less condense and not degraded in dying cells
- do not disrupt development
how do you determine the correct position of a gene for a recessive mutation (3)
- transform each gene into mutant and observe for complementation/WT phenotype
- sequence allele of all genes in mutant; correct gene will have a mutation
- obtain new loss of function mutants for each each gene; mutation in one of the genes should have similar phenotype to mutant phenotype
how do you make a genomic DNA library (3)
- extract DNA from cells and digest with restrictive enzyme
- DNA fragments inserted into cloning vectors
- transform vectors into bacterial cells; each cell will have a vector with different cDNA pieces
cDNA library
- represents cDNA copies of RNA
how to screen a DNA library (4)
- blot some bacterial cells on a membrane
- cell lysis, DNA denaturation and membrane fixing
- add labelled probes and incubate; wash away unhybridized probes
- identify colonies (will have hybridized DNA) that carry gene of interest
capsases (2)
- cell death executors
- first synthesized as an inactive protease precursor and is later activated by proteolysis
activation of caspases (2)
- self-activation: come together to form partial active sites
- by another cysteine protease/caspase
CED-4 and APAF-1 (2)
- evolutionarily conserved proteins involved in regulation of caspases
- have similar domains, reflecting comparable functions in C. elegans and humans
gain of function mutation (2)
- mutation that confers an enhanced activity on a protein
characteristics of gain of function mutations (2)
- increased protein activity
- uncontrolled or promiscuous activity
examples of uncontrolled or promiscuous activity (4)
- active without stimuli
- loss of expression control (wrong place, time, or amount)
- increase protein stability
- loss of negative regulation
in general, loss of function mutations are… (2)
- recessive
- half of normal product amount is enough for normal function
in general, gain of function mutations are… (2)
- dominant
- presence of the mutant version of the gene is enough to change phenotype
how do we assess the relationship between different mutants that function in the same pathway
- make double mutants and observe phenotype
interpret the statement: mutant 1 is epistatic to mutant 2
- mutant 1 suppresses defect in mutant 2; double mutant will have mutant 1 phenotype
- indicates that mutant 1 functions further downstream to mutant 2
how can you determine order in mutations with the same phenotype
- produce or search for gf mutation in one of the genes
- then make the double mutant
what are the 3 classes of protein functions in the conserved apoptotic family
- regulator
- adaptor
- effector
pathways initiating apoptosis (3)
- activation of cell death from inside the cell (intrinsic/mitochondrial pathway)
- activation of cell death from outside of cell (extrinsic/death receptor pathway)
- pathways intersect at effector caspases
intrinsic/mitochondrial pathway (5)
- mitochondria release cytochrome c
- cytochrome c activates caspase 9
- caspase cascade
- caspase cleavage of cellular proteins, nuclease activation
- death
