Lecture #31 - How do we find out what a gene does?

Question 1

Learning from mutants: phenotype to genotype

1. How can we work out what a gene mught do?
2. How can we make mutants?
3. What’s a Genetic Screen?

Answer 1

1. By studying organisms that are mutant for a particular gene
2. We can make mutants by treating organisms with radiation or chemicals.
3. Making and analysing lots of mutants is called a Genetic Screen. Genetic screens are designed to find mutations that affect a process or structure we are interested in (or metabolic pathway/drug interaction)

By studying the mutants we can learn how genes control that structure or process.

Question 2

Model Oragisms for Genetics

Name the 5 and why they used

Answer 2

1. Drosophila melanogaster -small and hv rapid life cycle
2. Mus musculus
3. Arabidopsis thaliana
4. Danio rerio
5. Caenorhabditis elegans

Used because they each have:

1. Share our genes
2. Fast life cycles
3. Lotsa offspring
4. Don’t take up too much space and resources

Split with mice 90mya and danio 300m. The plant used bc still informs abt hw organisms work

Humans - most of genes shared with mouse - conserved in same order

Question 3

Mutations are usedul in understanding development

1. So like, we can use mutants to study how embryos grow from a single fertilized egg (Development). But what does Development do?
2. What is this process?

Answer 3

1 - If mutated gene - can see how embryo affected

1. Development directs cells to become different types, sizes and shapes to form the structures of the body.
2. Genetically controlled

Question 4

So you know how naturally ocuring mutants are rare, genetic screen are therefore designed to…..

Answer 4

….accelerate the rate of mutations and increase phenotype discovery

Question 5

What’s the Heidelberg Screen?

Answer 5

What three scientists designed in Heidelberg. The idea was to find out all the genes that affect the development of a fruit fly (not just one or two - ALL)

Question 6

What happens in a normla Drosophilia during embryonic development?

Relate to genetic screens

Answer 6

Fertilised egg develops for a while inside the mother and then over time starts to segment - makes precursor segments that will make a head and thorax and legs etc. Everything after that is just interpretting the segments

How do we get to that stage?

They set up a very large mutant screen.

First accelerate rate of mutation and they decided to make point mutation (not targetted - we can treat flies with chemical and there will be mutations scross genome but we don’t know where).

• Feeding male fruit flies with chemical mutagen (ethyl methane sulfonate) - creates a male population that has mutations
• Crossed those males with females that were normal with known genetic back ground
• Every sperm set is carrying different set of mutations but they fed each fly so that they’d get around 14 mutations per fly (some might not hv effects so 14 guaranteed something important)
• Each male was mutated differently so the offspring will carry the different set of mutations of the father.
• Asterisks show mutation is present (tracker)
• The male off spring again crossed with normal females of known backgrounds
• Now both females and male off spring carry same set of mutations
• Now inbreed the sons and daughters (so both alleles damaged) aka same set of off spring inbred
• If sons and daughters were both heterozygous, 1/4 offspring will be homozygous and show the mutation
• Found that 139 genes involved in building the larva of a drosophila (saturation at 139 bc in those 580 homozygous, just kept getting repeats of the 139 genes)

Question 7

The Heidelberg screen: look for changes to the embryonic pattern

1. How?
2. Embryos with a defective cuticle…..
3. Thus….
4. What did they do to find all genes involved in segmentation?

Answer 7

1. Mutagenised flies, crossed to make homozygotes, checked the cuticle patterns of dead embryos.
2. Embryos with a defective cuticle have a mutation that affects segmentation.
3. Thus, every mutant represents a single gene involved in segmentation.
4. Kept doing this until all genes involved in segmentation were found

So each segment has polarity (brissel and smooth). Can get defective genes were all segments were smooth only (lose polarity?) or has genes that caused every 2nd segment to be gone

Question 8

Genetic Screens: the legacy

1. What’s positional cloning?
2. What is this similar to?
3. Similar (related) genes can be…..
4. E.g.

Answer 8

1. The mutant gene that causes the phenotype can be cloned by a process called ‘positional cloning’
2. This is similar to the procedure used to clone the cystic fibrosis gene
3. Similar (related) genes can be identified in other species, such as ourselves.
4. For example: similar genes to hedgehog are involved in vertebrate limb patterning and are implicated in causing disease e.g. colorectal cancer

Question 9

Genetic Screening summary

Answer 9

• Decide what process or structure you are interested in.
• Perform a genetic screen (w/ large no. of animals)
• Pick mutants that have defects in the thing you want to study
• Use these mutants to discover what the mutant genes normally do (study phenotype and then genotype)
• Using this technique you know the function of a gene (phenotype) before you know its sequence (genotype) - called forward genetics

Question 10

The mouse as a model organism

-Commonly used to test genes associated with genetic diseases

Name me 7 adv and 3 disadv

Answer 10

Adv:

• Mammalian- shares many biological functions with human
• Human and mouse genomes are the same size and many genes are in the same order (synteny).
• 99% of mouse genes have human homologues
• Similar immune system
• Small, easy to maintain, fast breeding

(8-10 per litter)

• Inbred lab strains reduce variation (use speicific strain mice so know that in the end, don’t end up with expected results)
• Good infrastructure and resources e.g. Jackson lab

Disadv:

• Mouse physiology can differ from human
• Still more expensive than other models (e.g. fish, flies but fish aint mammal so obvi can’t study them)
• Ethics concerns

Question 11

Transgenesis in mice

Answer 11

Put jellyfish into mice - this is a transgene

Basically adding foreign DNA into mice

Here, makes them flourescent - they don’t make the gene obvi

Question 12

This diagram is in handout

Answer 12

Question 13

What if you know the sequence of a gene, but not its function?

—->Knockout mice (5)

Answer 13

• We can remove (or replace) the gene we are interested in by genetically modifying an organism (genetic manipulation of stem cells)
• By examining the GM organism we should be able to work out what the gene normally does (phenotype)
• This process is often called “knocking-out” a gene.
• In mice the DNA of embryonic stem cells is manipulated to remove (null mutation) or replace a specific gene (more realistic). The stem cells are then inserted into a mouse blastocyst to make a chimaeric mouse.
• Knockout mice can be created for any gene provided you know the sequence of the gene first!

Question 14

Define Homologous recombination

Answer 14

When the targeted gene recombines with the targeting vector and is removed.

Question 15

Define Homologue

Answer 15

Gene that shares similar sequence and function.

Question 16

Define Knockout mouse

Answer 16

Mouse engineered (by homologous recombination) to lack a particular gene.

Question 17

Model organism

Answer 17

An animal used by geneticists to link genotype to phenotype, using reverse and forward genetic methods.

Question 18

Null

Answer 18

A non-functional gene allelle.

Question 19

Reverse genetics

Answer 19

When we disrupt the function of a known gene in order to work out what its purpose is (the opposite direction to forward genetics).

Question 20

Transgenic animal

Answer 20

Model organism that has been engineered to carry an extra piece of DNA or transgene by random integration.