model organisms and development 1

model organism

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

requirements for model organism

• mimics specific aspects of human biology

• is (comparatively) easy to work with

forward genetics

using phenotype (observable characteristic) to determine genotype (genetic makeup)

reverse genetics

using genotype (genetic makeup) to determine phenotype (observable characteristic)

eukaryotic model organisms

• S. cerevisiae (budding/baking/brewer’s yeast)

  • least similar to humans, but easiest to grow

• C. elegans (nematode)

• D. melanogaster (fruit fly)

• Danio rerio (zebrafish)

• Mus musculus (house mouse)

  • most similar to humans, but hardest to grow

S. cerevisiae (budding yeast)

• eukaryotic, unicellular fungus

• generation time: 2-3 hours

• can exist as haploid or diploid

• can reproduce sexually or asexually

• can be frozen and revived

generation time

time it takes to generate progeny that can then give rise to new progeny

S. cerevisiae life cycle

1. haploid organisms (either a or α mating type) bud, reproducing asexually and resulting in haploid progeny

2. two haploid organisms, one type a and one type α, can mate, reproducing sexually

3. diploid organisms (a/α) bud, reproducing asexually and resulting in diploid progeny

4. under starvation or stress conditions, a diploid organism can sporulate (undergo meiosis) and generate four haploid progeny (two type a, two type α)

C. elegans

• invertebrate animal, multicellular

• generation time: 3 days, 300 progeny

• extremely simple, translucent

  • can study on light microscope

• can trace the fate of each cell (1090 total)

  • invariant development

  • each individual has exactly the same number of cells, in the same pattern

• two sexes: male and hermaphrodite

  • can self-fertilize and be crossed

• can be frozen and revived

C. elegans life cycle

• under normal conditions, embryo → reproductive adult takes 60 hours

• under conditions of crowding, starvation, high temperature, individual enters dauer state and hibernates for several months before developing into reproductive adult

dauer

hibernation state of nematode larva

D. melanogaster

• invertebrate animal, multicellular

• generation time: 10 days, 100 progeny

• share 75% of human disease-causing genes

• body plan resembles human body plan (axes, organ systems)

• very well studied, many genetic tools

Danio rerio

• vertebrate animal, multicellular

• generation time: 203 months, 200 eggs

• optically translucent embryos and larvae

  • can study using light microscope

• relatively simple and inexpensive to maintain

• easily treated with small molecules for drug and toxicity screens

Mus musculus

• vertebrate animal

• generation time: 3 months, 2-12 pups

• small, easy to house

• commonly used to study human biology, perform preclinical testing

model organisms to study unique phenomena

• axolotl (A. mexicanum) → limb regeneration

• planaria (S. meditteranea and others) → whole-body regeneration

discovery of secretory pathway by Palade (1960s)

1. pulse-chase experiment

2. electron microscopy and autoradiography

3. model

pulse-chase experiment performed by Palade

• incubate slices of pancreatic tissue with radioactive leucine (pulse)

• then incubate with non-radioactive solution (chase)

• new proteins synthesized during the pulse have radioactive leucine

electron microscopy and autoradiography performed by Palade

• visualize which organelles contain proteins with radioactive leucine after different chases

• at different times after the chase was performed, proteins were in different organelles (ER, Golgi, secretory vesicles)

secretory pathway model proposed by Palade

• newly synthesized proteins move through organelles in a specific order

• ER → Golgi → secretory vesicles

basics of forward genetic screens

1. perturb lots of genes (randomly or systemically) (ex: chemical mutagen)

2. look for specific phenotype (organism dies, changes in some specific way)

3. figure out which gene was mutated

temperature sensitive mutations

• provide functional protein at permissive temperatures, but non-functional/less functional protein at restrictive temperatures

• often, single amino acid substitution within the hydrophobic core of a protein

identification of temperature sensitive sec mutants

• yeast mutagenized and incubated at two different temperatures (23°C and 36°C)

• mutant cells proliferate at permissive temperature, but not at restrictive temperature

• 87 mutants tested for activity of a secreted phosphatase via a colorimetric assay, only 1 did not have normal phosphatase secretion

  • sec1

sec1 mutant phosphatase secretion

• reduced secretion at restrictive temperature

• increased accumulation inside cells at restrictive temperature

mapping sec mutations onto secretory pathway

• total of ~50 sec mutants identified

• each had a defect at a specific point in the secretory pathway, identified using electron microscopy

• each fate of secretory proteins (secreted, which organelle they got stuck in) represents different Sec mutation

complementation in yeast

• if two sec mutations were in the same gene and haploid individuals (each with one mutation) were crossed, diploid would have temperature sensitive phenotype

• if two sec mutations were in different genes and haploid individuals (each with one mutation) were crossed, diploid would have wild type phenotype (normal secretion at restrictive temperature)

• narrowed down sec mutants to 23 individual genes

identifying mutant sec gene

• create yeast genomic library

• introduce different plasmids into yeast, test which plasmids rescue the mutant

• sequence plasmids that rescue to identify yeast gene responsible for phenotype

yeast genomic library production

1. cleave yeast double-stranded DNA with restriction nuclease

2. insert DNA fragments into plasmids

3. introduce plasmids into bacteria

determining function of sec gene products

• combine purified gene products with cell components in vitro and observe effect

• ex: Sar1, sec23, sec24, sec13, sec31 products + purified ER membrane + GTP → COPII vesicles

sec gene products in cell free (in vitro) system

produce a protein-coated vesicle

identifying human homologs of yeast genes

• create cDNA plasmids from human tissue

• introduce different cDNAs into yeast, test which plasmids rescue the mutant

• sequence cDNAs that rescue to identify human gene responsible for a phenotype

cranio-lenticulo-sutural dysplasia

• rare genetic disease characterized by improper collagen secretion causing altered skeletal development

• caused by phenylalanine → leucine substitution in Sec23A

• mutant Sec23A does not effective bind Sec13/31 coat → impaired secretion