Lecture 35

Question 1

what are model organisms

Answer 1

non human species used to study a specific biological phenomenon or disease

Question 2

why do we use model organisms

Answer 2

cant experiment on human so we need to learn from other organisms

Question 3

what are the characteristics of model organisms

Answer 3

mimic specific aspects of human biology
are somewhat easy to work with

Question 4

what are model organisms often used for

Answer 4

forward or reverse genetics

Question 5

what are reverse genetics

Answer 5

genotype –> phenotype
knockout mouse, role of gene on phenotype

Question 6

what are forward genetics

Answer 6

phenotypes to genotype
what genes regulate trait

Question 7

name and describe eukaryotic model organisms

Answer 7

s cerevisiae (easiest to grow, least like humans)
c elegans
d melanogaster
danio rerio
mus musculus (hardest to grow, most like humans= diverges from humans more recently in tree)

Question 8

Describe s cerevisiae

Answer 8

eukaryotic
unicellular fungus
generation time = 2-3 hrs (amount of time offspring becomes able to reproduce)
can exist as haploid or diploid
can reproduce sexually or asexually
can be frozen and revived

Question 9

describe life cycle of S. cerevisiae

Answer 9

haploid - asexual reproduction
haploid in stress conditions = easier to know effect of gene = easy to mutate since one copy
mates toeter and reproduces = diploid
helps undersatnd relationship between diff genes and diff copies of same genes and diff genes

Question 10

describe c elegans

Answer 10

invertebrate eukaryote
mutlicellular
generation time = 3 days, 300 progeny = many
extremely simple, translucent - helps understand development
can trace fate of each cell = 1090 total = invariant development tree = every adult made of same # of cells and develop in same exact way
two sexes = male and hermaphrodite - can be self fertilized and be crossed
can be frozen and revived

Question 11

describe life cycle of c elegans

Answer 11

embryo - 12 hrs later, not too long embryonic development, leads to full worm
if in conditions of crowding, starvation or high temp = leads to dauer = like quiescence then several months later leads to full worm

Question 12

describe d melanogaster

Answer 12

more similar to humans - similar cns, body plan, musculoskeletal aspects and digestive tract
invertebrate animal
mutilcellular
generation time = 10 days, 100 progeny
more complex than c elegans
share 75% of human disease causing genes
very well studied, many genetic tools

Question 13

describe danio rerio

Answer 13

vertebrate animal
multicellular
generation time = 2-3 months, 200 eggs
optically translucent embryos and larvae
relatively simple and inexpensive to maintain
easily treated with small molecules for drug and toxicity screens - easier since grown in water
closer to humans for biological purpsoes = melanocytes

Question 14

describe mus musculus

Answer 14

vertebrate mammal
generation time = 3 months, 2-12 pups
small easy to house for mammals
commonly used to study human biology, preform preclinical testing
mice are not always perfect models for humans, many differences stills

Question 15

describe 2 emerging model organisms to study unique phenomena

Answer 15

do things humans cant
axolotl = can regrow limbs
planaria = can regenerate everything, whole body of stem cells

Question 16

describe basics of forward genetic screens - 3

Answer 16

pheno –> geno
1 - pertrubs lots of genes - randomly or systemically
2 - look for specific phenotype - organism dies, changes in some specific way
3- figure out which gene mutated

Question 17

describe Finding cell division cycle (cdc) mutants in S. cerevisiae - gen

Answer 17

cell cycle easy to see in yeast
g1 s g2 m - look at stuck cells - phenotype

Question 18

describe temp sensitive mutations to study cell cycle

Answer 18

at permissive temps = grows normally
are restrictive temps = doesnt fold properly and leads to mutant

Question 19

describe replica plating

Answer 19

to isolate temp sensitive mutants
add mutagen to yeast, master plate, use sticky sterile velveteen, imprint colonies and test at diff temps
at high temp = mutants dont grow

Question 20

WHAT DID they find from cdc mutants - 3 diff mutants

Answer 20

cdc1 mutant = never keeps going, frozen in g1
cdc2 mutant =form bud but freeze not far in, frozen in s
cdc3 mutant = cytokineses cannot happen, late stage mitosis affected

Question 21

what do cdc mutants correspond to

Answer 21

cell cycle genes and other stuff
cdc1 = make cell wall
cdc2 = dna pol
cdc3 = septin - important for cytokinesis

Question 22

what can yeast be used to preform and explain

Answer 22

Complementation
used to test if 2 mutant strains have mutations in same gene
should be fine if one has wild type of each gene - if not on same gene

Question 23

how to identify mutant genes - 4 main aspects

Answer 23

make genetic library and does rescue experiments
1 - introduce diff plasmids into yeast, test which plasmids rescue mutant
2 - Sequence plasmids that rescue to identify yeast gene responsible for phenotype
3 - introduce diff cdnas into yeast and test which rescue mutant
4 - sequence cdnas that rescue to identify human gene responsible for a phenotype

Question 24

describe identifying the human version of cdc28

Answer 24

use mutant and human cdna library, extract and sequence plasmids = colonies survived are rescued
cdc 2 (s pombe) = cdc28 (s cerevisiae) = cdk1 (human) - very conserved

Question 25

what does c elegans undergo

Answer 25

invariant embryogenesis

Question 26

describe invariant embryogenesis of c elegans

Answer 26

every embryo develops with same pattern of cell divisions and migrations
adult worms have exactly 959 cells, generated in exact same way
useful when performing genetic screens to understand development

Question 27

describe what happens in c elegans embryo

Answer 27

1090 cells form during embryogenesis
959 cells present in adult
131 cells die during embryogenesis
dying cells engulfed quick - too hard to see so mutant that alters phenotype

Question 28

describe discovery of cell death mutants in c elegans - ced 1

Answer 28

ced 1 mutant = cells still die but cannot be engulfed
dying cell corpses very easy to see

Question 29

describe discovery of cell death mutants in c elegans - ced 1 ced 3

Answer 29

supressor screen to find mutants in the ced 1 background
ced 1; ced 3 double mutants no longer can visble cell corpses -ced3 essential for cell death
Conclusion = cell death is an active process

Question 30

describe egl-1 mutants

Answer 30

have too much cell death
cannot lay eggs = no hsns (hermaphrodite specific neurons)
needed for laying eggs
make them but too much cell death and they die = these cells more sensitive to death signal
egl 1-ced3 double mutants have hsns
supressor screens with egl1 led to discovery of ced4 (egl 1 mutants also have hsns when ced 4 mutated), ced 4 rescues excessive cell death

Question 31

describe ced 9 mutants

Answer 31

Excessive cell death - many cells die
if ced 9 on = cell survives
simialr sequence to human bcl 2

Question 32

describe human bcl-2 in c elegans

Answer 32

can modulate death in c elegance
heat shock = promoter activated
bcl 2 = many extra cells
ced 9 - after heat shcok, now have many extra cells

Question 33

what we know know about cell death - 4

Answer 33

1 - normally, bcl 2 inhibits BAX and BAK (binds and sequesters)
2 - in death conditions, BAK and BAK permeabilize the mito membrane (oligomerizes and forms pore)
3 - mitochondrial cytochrome c is released which activates caspase 9 (via apaf 1)
4 - capase 9 cleaves and activates caspase 3, causing irrev death

Question 34

describe ced 9 - human equiv and loss

Answer 34

c elegans ced9 = human BCL2
loss of ced9 causes more death, makes pores all over place

Question 35

describe ced 4 - human equiv and loss

Answer 35

c elegans ced4= human apaf1
loss of ced4 causes less death
Cannot activate cascade

Question 36

describe ced 3 - human equiv and loss

Answer 36

c elegans ced 3 = human caspase 3
loss of ced 3 causes less death
no chewing up so less death

Question 37

describe egl 1 - effect on cell death

Answer 37

egl 1 inhibits ced9 and original mutation was a gain of function =
more egl 1 activity causes more death