pastor lecture 1 Flashcards
what do we use model organisms for?
-Testing compounds
* Genetic models- You have a gene, and you want to know what it does (reverse genetics)
* Genetic screens- You want to find genes whose loss or gain produces a phenotype (forward genetics)
What are some of the eukaryotic model organsisms that we use?
-s cervisiae, C.elegans, D melanogaster (fruit fly), danio rerio (zebrafish), mus musculus (house mouse)
What is the divergence of humans and some model organisms (Photo)
What is S cerevisiae?
What is its generation time?
What can it exist as?
-eukaryotic, unicellular fungi
* Generation time, 2-3 hours
* Can exist as haploid or diploid
How can S cerevsia reproduce?
How can be stored?
Why do bud scars occur/
-Can reproduce asexually or
sexually
* Can be frozen and revived
-as yeast get older they have these scars which indicate how mnay yeast have bud from it
What do you not need for yeast?
What can you do with them?
-dont need a sophisticated crosses for yeast, but you can cross them to make a diploid organism to check for dominant/recessive mutation
What are C.elegans?
What is their generation time?
What is special about them?
-animal, multicellular
* Generation time: 3 days, 300 progeny
* Extremely simple, translucent, can
trace fate of each (1090 total) cell
What are the sexes for C. elegans, and how do they reproduce?
How are they stored?
How do you get a double mutant?
-Two sexes, male and hermaphrodite.
* Can self fertilize, can be crossed
* Can be frozen and revived
-can cross male and with a hemaprodite to get a double mutant
What happens to C.elegans when there is no food/crowding in development?
-in absence of nutrients they can end up in a dauer state and can be frozen to be preserved indefinetly (since they are in a suspended state
What are D melanogaster?
What is their generation time?
What are they more complex than?
how well are they studied?
Animal, multicellular
* Generation time: 10 days, >100
progeny
* More complex than C elegans
* Extremely well studied, numerous
genetic tools
What is the danio rerio (zebra fish)?
What is its generation time
-what type of development?
What is special about their eggs
What can you target genes with?
Vertebrate animal
* Generation time: 2-3 months, 200- 300 eggs
* Mid-development relatively similar
to mammal
* Eggs are transparent
* Can target genes with morpholinos
What happens if you inject morpholino into zebrafish?
How can you watch it?
-morpholino has an alternative backbone to DNA, but when injected into zebrafish that is reverse complimentary to zebrafish egg and watch it grow
-can watch in real time and much quicker to complete than in mice
Where is there the most conservation between vertebrates?
in mid embryogensis
What is generation time of mus musculus?
How big are they?
What type of strains are availble?
What makes them advantageous
Generation time: 3 months, 2 – 12
progeny
* Very small as mammals go, easy to house.
* Inbred strains available
* Closer to humans than most mammals
What are the larger mammalian models?
What are the advantages to using each?
-rattus norvegicus ( larger size better for toxicology 150g vs 20g) metabolism is more human like
-marmoset and rhesus are closer to humans bu have long generation time, small litter size and are expensive
what is the arabidopsis thaliana?
What is its generation time?
How do they generate progeny?
What can you do with their seeds?
Model organism of the plant world.
* Short generation time (~2 months)
* Normally self-fertilize, but can be crossed
* Dry seeds can be easily stored and shipped
what can axolotls do?
What research are armadillos used for?
-can be used to regenerate limbs
-human leprocy research works with armadillos
What can you make stem cells from?
-adult cells and blastocysts
Where are organoids found?
What do paneth cells secrete?
-in the intestinal epithelium
-secrete molecules to allow cells survival
What are the 3 steps for the basics of screening?
Step 1: perturb lots of genes (randomly or systemically)
* Most common, mutate genes
Step 2: look for a phenotype
* Organisms dies, changes etc. in some obvious way
Step 3: Figure out what gene you mutated
What do you stamp mutated S. cerevisiae when doing screening/replica painting?
What do you do after the mutant has been stamped?
-physically stamp the mutated yeast plate on sterile velveteen then stamp the velveteen onto various plates with diff medias to see which one grows
What can temperature sensitive mutants do?
What is the lower temperature called?
What happens at high temps?
-temp. sensitive mutants can destabilize the protein
-low temperature is known as permissive temp.
-protein denatures and unfolds
When do all colonies survive when screening for temp sensitive mutants?
-survive at low temp
How do you find cell cycle mutants (cdc)
-transferred yeast from permissive (23C) to restrictive (36C) temperature,
looked for mutants that paused
at one point in the cell cycle
What happens in cdc2 vs cdc3 mutant yeast
in cdc2 mutant the cells dont replicate
-cdc3 they undergo multiple cycles of division without budding off
-after 3-5 cycles they explode
What type of mutation does cdc1, cdc 2, cdc 3 cause?
-cdc-1 is involved in cell wall synthesis (mutant cant form buds)
-cdc-2 is a dna pol mutant
-cdc 3 is a cytokinesis mutant
What can S cerevisiae survive as and be used for?
-can mate, survive as diploid and be used for complementation
How can you identify the mutant gene using yeast cDNA?
What are you sequencing/checking for
-can sequence the plasmid that is responsible (the plasmid that is overlapping with the genome)
-can check to see if WT rescues genome
How was human cdc28 found, what are the steps that were done?
-you use cdc2ts mutant S. pombe yeast and then a human cDNA library (cDNA in plasmids)
-then cdc2 S.pombe expresses random human genes at high temps
-then extract and sequence the plasmid
What can substitute for S.pombe?
-human CDK1 can sub for S pombe (cyclin dependent kinase)
What forms in a predictable fashion for C.elegans?
-the 1090 cells form in a predictable fashion
What do you have a welath of with C.elegans?
What do you know will form?
-what can you tell has failled to form?
-what can be complemented?
You have a wealth of mutant worms with defined phenotypes (unc,
lin)
* You know exactly what cells are supposed to form, exactly when.
* You can look at any mutant and determine what cells failed to form.
* You can complement these mutants with cDNA or genomic DNA, then figure out the sequence of the gene
