chapter 14 Flashcards
ecoli
- The best studied prokaryote
- Generation time: 20 minutes (shortest of prokaryotic organisms)
- Genome size: 4.64 Mb
- Number of chromosomes: 1 (+ plasmids)
- Estimated protein coding genes: 4262
- Advanced our understanding of mechanisms behind DNA replication, transcription, translation
- One of the easiest life forms to maintain in the laboratory
S. cerevisiae/bakers yeast
- Generation time: 2-3 hours
- Genome size: 12.2 Mb (3 times ecoli)
- Number of chromosomes: 16 (2n = 32)
- Estimated protein coding genes: 6728
- Can be cultivated as easily as E. coli, yet represents a eukaryotic cell with compartmentalized organelles like multicellular life forms
- Used for thousands of years for baking and fermenting alcohol!
C. elegans
- Nematode worm
- Generation time: 3 days
- Genome size: 103 Mb
- Number of chromosomes: 5 (2n = 10)
- Estimated protein coding genes: 20,452
- Arguably one of the simplest multicellular life forms
- First animal to have its nervous system (connectome mapped)
- ~ 300 neurons
D. melanogaster
Fruit fly/vinegar fly
* Generation time: 2 weeks
* Genome size: 169 Mb
* Number of chromosomes: 4 (2n = 8)
* Estimated protein coding genes: 14,217
* Greatest model organism for genetics
* Advanced our understanding of autosomal inheritance, sex-linked
inheritance, genetic mapping, and so much more
* Many neuronal circuits characterized for courtship, aggression, feeding, memory/learning, sensory systems, etc.
* Estimated to have between 100 000 and 200 000 neurons total
M. musculus
- House mouse
- Generation time: 10 weeks
- Genome size: 2731 Mb
- Number of chromosomes: 20 (2n = 40)
- Estimated protein coding genes: 22,322
- The best studied mammal
- Like Drosophila, the mouse has many tools for genetic and neurological manipulation
- Closest relative to humans as far as major model organisms go
- Estimated to have ~ 71 000 000 neurons
-closer to humans, harder experiments
when to use E. coli or S. cerevisiae
E. coli or S. cerevisiae are best if you are only concerned with studying
cellular processes
when to use C. elegans
best if you are only concerned with understanding the
physiological patterns of neuronal circuits for simple behaviours
when to use Drosophila
Drosophila is a complex multicellular animal with complex anatomical
systems, yet has a short generation time and is a versatile model system
when to use mice
Mice have physiology closest to humans and studies on mice offer more relevance to understanding complex systems in humans like the brain
Forward Genetics
The identification of genes by mutant phenotypes. identifies genes responsible for a specific phenotype by screening for mutation. starts with a phenotype (e.g., wingless fly) and tries to identify the gene(s) responsible.
- E.g. the discovery of the white gene in Drosophila
- Breed individuals exposed to mutagens to wild-type individuals
- Screen offspring for mutations through selective breeding
Imagine you are performing a forward genetic screen on fruit
flies… Mutagenesis Strategies
Dominant
-Expose males to mutagen and mate them to wild-type females
-If mutation is dominant, the process is much easier
-Dominant mutations appear in F1 progeny
-Can collect multiple mutants and generate pure breeding stock
-Generally dominant mutations are more rare
Recessive
-If mutation is recessive, the process is harder
-Isolate multiple F1 progeny and mate them to wild-type individuals
-Collect a bunch of F2 progeny and interbreed a bunch of them
-Identify recessive mutants in F3 generation and collect multiple to begin a pure-breeding stock
-Generally recessive mutations are more common
-If mutation is recessive, the process is much easier in organisms that can self-fertilize
-This is limited to specific model organisms
-Not possible in Drosophila
(cant figure out what flies are carries and which arent so you have to mate single males and females to control it)
Drosophila mutagenesis: X-linked Mutations
Attached-X mutants are a great tool if an X-linked mutant is generated
- Attached-X are phenotypically female, but their X chromosomes
are inherited together
-Enables X-linked male mutants to pass their X chromosome directly to
male offspring (none of male offspring will have mutated X)
Females: Either give both X chromosomes (XX) to the egg
Or give no X at all (O) — which is nonviable unless a Y is also present
look at slide for this
Determining Number of Mutated Genes in Forward Screen
Complementation test!
Remember:
* If crossing two recessive mutants produces wild-type progeny, there are two separate genes which are mutated. the two mutations are in different genes → complementation occurred
* If crossing two recessive mutants produces mutant progeny, there is only a single mutated gene. The mutations are in the same gene → no complementation
Let’s say you have two mutant flies that are both wingless.
Cross mutant A × mutant B
If offspring have normal wings, it means:
A and B had mutations in different genes
Their functional copies from the other parent “complement” the defect
If offspring are still wingless:
A and B have mutations in the same gene
No functional copy present = mutant phenotype persists
Reverse Genetics
Begins with a gene of interest and manipulates the gene to identify mutant phenotypes
with large genomic sequences available, reverse genetics is
a more attractive approach
Common approaches include:
* Mutagenesis through gene editing
* Gene knockout
* Gene knockdown
* Reporter genes
Reverse genetics: CRISPR-Cas9 Gene Editing
Cas9: a nuclease that makes double-stranded breaks - requires guide RNA that is complementary to genomic target (choose where it cuts, suring guide RNA complementary to target site, select where to cut and knockout target region)
* Multiple guide RNAs can be used to make multiple cuts
* Cuts repaired by cell through either NHEJ or homologous recombination (SDSA)
To edit DNA, inject embryo with:
* Engineered guide RNA
* Cas9 mRNA
* Donor DNA template
Reverse genetics: Gene Knockout
Deletion of the coding region of a gene
- Easily done through CRISPR
- Induce double stranded break at
target gene - Supply a template containing a
selectable marker that alters phenotype of organism - Selectable marker replaces deleted
coding sequence, and allows confirmation of knockout
Reverse genetics: Gene Knockdown
RNA interference (RNAi) can be used for silencing a gene of interest. It’s especially useful when deleting the gene would be lethal or too severe
- Insert gene that produces RNA that can complementary base pair with matching mRNA of interest
- If this inserted gene is expressed in high amounts, a gene of interest will be silenced through degradation of mRNA
-insert gene into genome that will encode double stranded RNA, will inhibit gene of interest-> select gene you want inhibited and knock it down instead of deleting it
Reverse genetics: Reporter Genes
A gene can act as a reporter gene if its protein product can be
detected directly or indirectly (responds where gene is expressed) - studied in multicellular organisms to determine what tissue the gene of interest plays a role in
- For example, green fluorescent protein (GFP) is a very commonly used reporter gene (stimulated at different wavelengths, different fluorescent colours, looking at different reporters at once in different places at once)
- The lacZ gene (beta galactosidase) is another example. X-gal: a chemical mimicking lactose. Produces a blue colour when degraded by beta galactosidase
- Often used in microscopic life forms
Reporter Genes are Often Fusion Genes: A fusion gene is made by joining two genes together — in this case, the reporter gene is fused to your gene of interest so they’re regulated or translated together.
Transcriptional Fusion
Reporter gene (e.g. GFP) is inserted right after the transcription start site
It is under the same promoter as the gene of interest
But the reporter is translated on its own, not fused to the original protein. Presence of GFP indicates where gene is transcribed
Translational Fusion
The reporter gene is inserted in-frame within or at the end of the coding sequence. GFP tagged onto
translated protein.
It becomes part of the same open reading frame (ORF) and is translated as part of the protein. What it tells you:
➡️ Where the protein goes in the cell
➡️ Can be used to track protein
Enhancer Trapping
inserting a transgene with a weak promoter in a genome, The promoter is too weak to express the transgene at significant amounts
- However if the gene is inserted near an endogenous enhancer, the gene could be expressed at high amounts and with specific expression patterns
GAL4-UAS System – an Enhancer Trap
Flies don’t naturally express GAL4 or respond to UAS, so you get no background expression unless you add them.
This enables a system where GAL4 expression can be controlled by a specific promoter (driver) and a gene of interest can be inserted under the regulation of UAS (responder
GAL4-UAS Transgenic System
There are thousands of transgenic fly
lines that have either:
- GAL4 sequence fused with a
promoter sequence (driver line) - There are thousands of different driver lines. Many tools to force expression of GAL4 in specific Drosophila tissues - E.g. specific organs, specific neurons, specific localized cells
- UAS sequence fused with a target
gene sequence (responder line) - There are thousands of different responder lines, Many reporter tools,
Many tools to manipulate gene expression, Many tools to manipulate neurons
Custom driver lines and responder lines can be made via CRISPR,
and mixed and matched with existing driver and responder lines
The GAL4-UAS system is only
active when a driver line fly is
crossed to a responder line fly (driver line or responder line on its
own does not show expression of any
of these transgenes)
GAL4-UAS as reporter system
-Reporter system to monitor where a gene of interest is expressed
Promoter sequence fused to GAL4 can be any promoter
* Based on where driver is located: Can be specific – e.g promoter of a specific neuron type or * Can be general/broad – e.g promoter of actin gene
* Leads to expression of GFP only in specific neuron
* Leads to expression of GFP in every cell of fly
* Useful for finding where a gene of interest is expresses
GAL4-UAS and RNAi Gene knockdown system
Gene knockdown system by using GAL4 to express RNAi construct
-All offspring with driver and responder will have the RNAi construct expressed in all cells
* RNAi construct will bind to specific mRNA of interest, preventing protein synthesis
* Can also be more restricted by controlling GAL4 through a different promoter (e.g. expressing
RNAi construct in specific neurons)
Driver line Responder line (ex: inhibit pheromone production in oenocytes)
Over-expression system by using GAL4
Over-expression system by using GAL4 to express an endogenous gene at higher amounts
offspring with driver and responder will express gene of interest
in all cells in addition to wherever that gene is endogenously expressed
* Doubles gene expression
* Can express gene in specific tissue using a more restrictive promoter
(e.g in certain neurons, in specific muscle tissue, reproductive tissue, etc.
(ex:upregulate pheromone genes in oenocytes - want to force this expression/double it)
