Genetics Notes

Learning Outcomes

• Genetics techniques and phenomena can be applied to shed new light on biological questions.
• A receptor for a specific odor was identified using genetic methods, from mutation to mapping to gene identification.
• Regulation of gene expression could be analyzed by a combination of molecular biology and genetics.
• Model systems are important for understanding human biology, such as olfaction.
• Understand the molecular and neuronal basis of olfaction.
• There are still vast gaps in our knowledge of biology.

Model Systems

• Experiments can be performed that are not possible in humans or other organisms.
• There is a lot of infrastructure including experimental tools and background knowledge.
• Fundamental principles can be gained.
• Conserved genes and pathways can be identified and studied.

Historical Importance

• Gene structure and genetic code were discovered using T4 bacteriophage.
• Transcriptional regulation was discovered using E. coli.
• Secretory machinery was discovered using yeast.

Understanding Smell

• Detect chemical environment to detect prey, predators, and toxins.
• Has effects on physiology; for example, the smell of food affects insulin secretion.
• Important for enjoying food.
• Diseases can impair smell, which affects diet and quality of life, and can be a sign of neurodegeneration.

Caenorhabditis elegans

• Nematode (Roundworm) aka "the worm".
• Small (adult ~1mm).
• Found in soil and rotting fruit worldwide.
• Free-living (many parasitic relatives).
• Has neurons, muscle, intestine, and skin.

C. elegans: Useful Features

• Simple.
• Transparent.
• Stereotyped anatomy.
• Fast life cycle (3-5 days).
• Small and cheap to culture.
• Self-fertilizing hermaphrodites.

C. elegans Can Smell

• Worms move to attractive odor.

Worms are Attracted to Diacetyl

• Diacetyl is the "buttery" flavor in popcorn.
  • Chemical formula: CH3COCOCH3

The Nose of the Worm

• Several olfactory neurons: AWA, AWB, AWC.
• Each neuron is unique and identifiable.
• Sensory neurons send out neurites with access to environment.

Identifying the Function of Single Neurons

• Determine the function of neuron by testing the effects of removing that neuron.
• In worms, neurons and other cells can be killed with a laser.
  • e.g., Fire laser at AWA neurons to kill them.
• Test behavior of animals that don’t have certain neurons.
  • e.g., Are animals without AWA neurons still attracted to diacetyl?

AWA Neurons Sense Diacetyl

• But retain attraction to other chemicals e.g., benzaldehyde.
• AWA present: Worms attracted to diacetyl.
• AWA killed with a laser: Worms no longer attracted to diacetyl, but still attracted to benzaldehyde.

Summary: Background on Worm Olfaction

• Worms can smell.
• Behavioral response: move toward attractive compounds.
• Diacetyl attracts worms.
• AWA and other sensory neurons are:
  • uniquely identifiable
  • have nerve endings exposed to environment
• AWA neurons sense the chemical diacetyl.
• Like humans, worms have sensory neurons that can detect smells.
• In worms and humans, the information from the smells is conveyed through the nervous system to influence behaviour.

What are the Genes that Mediate Smell?

Look for Mutants that Cannot Smell

• Used mutagens to generate random mutations.
• Screened through population of random mutants for animals that cannot sense diacetyl.
• Found many recessive mutants.
• Complementation analysis: determined which alleles affect the same gene.
• Focused on one gene: odr-10
  • Mutant - can’t sense diacetyl

odr-10 Mutants Cannot Smell Diacetyl

• Can sense other chemicals.
• Defect is specific to diacetyl.
• Not a general inability to smell.
• Wild type: Attracted to diacetyl.
• odr-10 mutant: No attraction to diacetyl.

Removing a Gene by Mutation Reveals its Function

• Analogy:
  • No wheels, can’t move.
  • Therefore, wheels required for car to move.
• Remove odr-10 by mutation
  • No odr-10, can’t smell diacetyl
  • Therefore odr-10 is required for smelling diacetyl

Using Recombination Mapping to Locate a Gene

• Map odr-10 by linkage to a chromosome
• Finer mapping to a small region of the chromosome
• Follow the gene by its phenotype
• LG X sup-7stP33 odr-10 unc-6

Using Rescue to Identify odr-10

• Rescue the odr-10 phenotype
  • Make transgenic animals with different pieces of wild type DNA
  • Experiment: which piece(s) rescue?
  • Using smaller pieces to narrow down region
• Inference
  • One of these genes might be odr-10
  • Gene 4 is a candidate for odr-10

Sequence DNA to Confirm odr-10 Gene Identity

• Sequence the rescuing region: infer gene structure
• Rescuing genomic DNA: ~3.3kb (promoter and coding sequence with 8 exons)
• mRNA: 1118bp
• Confirm different alleles have different mutations in the same gene
• ky32 point mutation CAC (His) > TAC (Tyr)
• ATG (Start) TGA (stop)
• ky225 deletion

Protein Structure of ODR-10

• odr-10 encodes a 7-transmembrane receptor (339 amino acids)
• Large family of receptors
• These receptors lie on the plasma membrane and transmit signals into the cell.
  • e.g., presence of odors

Summary: Applying Genetic Tools to Identify a Gene and its Function

• Identify a gene by screening random mutations for a specific phenotype.
• Removing a gene by mutation allows one to infer its function.
• Locate a gene by recombination mapping and rescue
• Sequence the gene to find what protein it encodes.
• Locate mutations to confirm gene identification.
• LG X sup-7stP33 odr-10 unc-6

Using Reporter Genes to Identify Where odr-10 is Expressed

• Construct plasmid: attach green fluorescent protein (GFP) to the odr-10 promoter
  • Drive GFP under control of odr-10 promoter
  • Transform worms with this plasmid
• Natural odr-10 gene: Promoter - coding sequence
• Man-made odr-10::GFP: Promoter odr-10 - GFP

Gene Expression: odr-10 is Expressed in Olfactory Neurons

• odr-10::GFP is found in AWA neurons, which senses diacetyl
• odr-10 is required for sensing diacetyl
• odr-10 acts in AWA to sense diacetyl

How Does odr-10 Work With Other Genes Needed for Smell?

• odr-7 mutants also can’t sense diacetyl, just like odr-10 mutants
• The odr-7 gene encodes a transcription factor
• How does odr-7 work with odr-10 to enable worms to sense diacetyl?

Gene Regulation: odr-7 Switches on odr-10 Expression

• Hypothesis: Since odr-7 encodes a transcription factor, maybe odr-7 mutants can’t smell because they don’t express odr-10 in AWA neurons
• Test hypothesis: Use the odr-10::GFP reporter to see what happens in the odr-7 mutant
• Result: In the odr-7 mutant, odr-10::GFP is not expressed
• Conclusion: the odr-7 transcription factor is needed to switch on odr-10 expression in AWA neurons

Summary: Gene Expression and Regulation

• Genes need to be in the right cell to function properly
• Generate reporter genes to determine where a gene is expressed.
• Understand gene regulation by examining gene expression in a mutant.

Understanding Mammalian Biology from Worm Genetics

• 7-transmembrane proteins similar to odr-10 are present in mammals.
• Thought to be olfactory receptors
  • large family
  • expressed in olfactory epithelium
• But actual function not tested

Understanding Mammalian Biology from Worm Genetics

• No function demonstrated for candidate receptors in mammals
  • Chemicals that they detect unknown
  • No direct evidence for role in olfaction
• Genetic data: odr-10 is the receptor for diacetyl
• First functional evidence that show this family of receptors are involved in olfaction

What We Learnt About the Biology of Smell

• 7-transmembrane receptors like ODR-10 sense odours in olfactory neurons
• Different receptors sense different odours
• Some odours can activate more than one receptor
• Some receptors can sense more than one odour

We Still Don’t Know Everything

• How is information about smells transmitted in the nervous system?
• How do we represent different smells in the brain?
• Like olfaction, many areas of biology are still full of unknowns and are active areas of research.

Using Genetics to Understand Biology

• Smell - an example of important biology
  • Identify a gene by screening for mutations, complementation analysis, recombination mapping, rescue experiments and DNA sequencing
  • Determine where a gene is expressed by constructing plasmids that report expression
  • Understanding gene regulation
• Experiments are difficult or expensive in humans and mammals
  • Simple model systems can reveal information about conserved genes rapidly and in a cost-effective way
  • Applies to most areas of biology