20250214_BIOC212_Model organisms & Dev 1

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• Model Organisms & Development

  • Course Code: ANAT212/BIOC212

  • Date: February 14, 2025

  • Instructor: Katie Cockburn, PhD, Assistant Professor, Dept. of Biochemistry

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• Outline of Model Organisms & Development Part I

  • Brief overview of model organisms and their uses

  • Study of S. cerevisiae and identification of vesicular trafficking machinery

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• Importance of Using Model Organisms

  • A model organism is a non-human species used to study biological phenomena or diseases.

  • Justification for use:

    1. Mimic specific aspects of human biology

    2. Generally easier to work with compared to human subjects

  • Common applications include forward and reverse genetics

    • Phenotype: observable characteristics

    • Genotype: genetic makeup

    • Forward Genetics: studying traits to identify the genes responsible.

    • Reverse Genetics: manipulating a gene to observe phenotypic effects.

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• Eukaryotic Model Organisms

  • S. cerevisiae (budding yeast): Used in baking and brewing

  • C. elegans (nematode): A simple roundworm for genetic studies

  • D. melanogaster (fruit fly): Crucial for genetic studies

  • Danio rerio (zebrafish): A vertebrate model organism

  • Mus musculus (house mouse): Mammalian model for advanced studies

  • General characteristics: easier to grow, relevant biological insights for humans

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• Divergence of Humans & Common Model Organisms

  • Timeline of divergence from the last common ancestor (in millions of years)

  • Reference: Zaidel-Bar et al, JCB (2009)

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• S. cerevisiae:

  • Eukaryotic unicellular fungus

  • Generation Time: 2 to 3 hours

  • Can exist as haploid or diploid forms; reproduces both sexually and asexually

  • Capable of being frozen and revived

  • Reference: Bernstein & Bernstein (2020)

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• Life Cycle of S. cerevisiae:

  • Diagram showing haploid and diploid phases

  • Mating: between haploid types a and α

  • Processes include sporulation and budding

  • Reference: Wang et al, PLoS Computational Biology (2017)

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• C. elegans:

  • Invertebrate multicellular organism

  • Generation Time: 3 days (produces 300 progeny)

  • Features: simple and translucent body structure; can trace the fate of each of its 1090 cells

  • Reproduction: male and hermaphrodite; self-fertilization possible

  • Capable of freezing and reviving

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• Life Cycle of C. elegans:

  • Development involves specific durations for stages (14h, 12h, 8h, etc.) under varying conditions (crowding, starvation, temperature)

  • Reference: Images from Worm Atlas

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• D. melanogaster:

  • Invertebrate multicellular organism

  • Generation Time: 10 days (producing around 100 progeny)

  • Features: shares approximately 75% of genes linked to human diseases; extensively studied with many available genetic tools

  • Reference: Images from Droso4schools

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• Danio rerio:

  • A vertebrate, multicellular organism

  • Generation Time: 2-3 months (producing 200 eggs)

  • Benefits: embryos and larvae are optically translucent; simple and inexpensive maintenance; suitable for drug and toxicity screening

  • Reference: White et al. Nature Reviews Cancer (2013)

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• Mus musculus:

  • Vertebrate mammal model

  • Generation Time: 3 months (producing 2-12 pups)

  • Advantages: small, easy to house; key in studying human biology and preclinical testing

  • Caveats: not always an accurate model for human conditions

  • Reference: Delage et al. Pharmaceutics (2021)

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• Emerging Model Organisms:

  • Planaria (A. mexicanum): Known for limb regeneration

  • Planaria (S. meditteranea & others): Capable of regenerating entire bodies

  • Reference: Wells et al. eLife (2021); Beanelab.org

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• Outline Continuation: Model Organisms & Development Part I

  • Brief overview of model organisms and their uses

  • Focused study on S. cerevisiae and identification of vesicular trafficking machinery

  • Nobel Prize Recipients (2013):

    • Randy Schekman: COPII vesicle formation

    • Thomas Sudhof: Synaptic vesicle fusion

    • James Rothman: COPI & SNAREs

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• Discovery of the Secretory Pathway:

  • Conducted by George Palade in the 1960s

  • Pulse-chase experiment:

    1. Radioactive leucine incubation followed by non-radioactive solution

    2. Electron microscopy used to visualize where proteins are located within organelles

    3. Model established for the movement of proteins through ER → Golgi → secretory vesicles

  • Queries regarding the proteins that facilitate cargo movement between organelles.

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• Basics of Forward Genetic Screens:

  1. Perturb multiple genes (randomly/systemically), e.g., using chemical mutagens

  2. Search for specific phenotypes that emerge:

     • Organism shows death or distinct changes

  3. Identify the mutated gene responsible

  • Connection between phenotype and genotype

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• Temperature Sensitive Mutations:

  • Essential genes for secretory pathways; mutations often lead to cell death or growth defects

  • Mutations may involve single amino acid substitutions affecting protein function

  • Description of permissive and restrictive temperatures (23°C vs 37°C)

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• Identification of Temperature Sensitive Sec Mutants:

  • Screening led to identification of 1600 colonies, yielding 87 temperature sensitive (Ts) mutants

  • Tested phosphatase activities, revealing normal secretion in 86 mutants, but 1 mutant (sec1) showed defective secretion.

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• Mapping Sec Mutations:

  • Additional screens identified ~50 sec mutants, each with defects at specific points in the secretory pathway, classified via electron microscopy

  • Comparisons made between wildtype and sec4 mutants at different temperatures.

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• Complementation Tests in Yeast:

  • Used to determine if two mutant strains share mutations in the same gene

  • Resulted in narrowing down 50 sec mutants to 23 individual genes using complementation analysis.

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• Yeast Double-Stranded DNA Techniques:

  • Creation of yeast genomic DNA libraries through restriction enzyme cleavage

  • Introduction of plasmids into yeast to observe phenotypic rescue in mutant strains and sequence to identify responsible genes.

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• Determining Functions of Sec Gene Products:

  • Focus on SEC gene product purification and in vitro systems to demonstrate vesicle formation

  • Products like Sar1, Sec23, and others involved in COPII vesicle formation.

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• Identifying Human Homologs of Yeast Genes:

  • Introduction of human cDNAs into yeast models to rescue specific mutant phenotypes

  • Sequencing successful plasmids to identify corresponding human genes.

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• Identifying Human Versions of Sec Genes:

  • Characterization of sec23ts mutant's interaction with a cDNA library to find corresponding human homologs (Sec23A & Sec23B).

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• Implications of Secretion Machinery in Human Disease:

  • Specific mutation (phenylalanine to leucine) in Sec23A linked to cranio-lenticulo-sutural dysplasia

  • Disease characterized by improper collagen secretion, affecting skeletal development.

  • Mutant Sec23A shows compromised interaction with critical coat proteins (Sec13/31), leading to impaired secretion.