Dissection of Gene Function part II: Reverse Genetics and Mutant Analysis

Question 1

How do we get from a mutation to the gene? 3

Answer 1

• Mapping:
• Genome sequencing:
• Transposon tagging:

Question 2

How do we get from a mutation to the gene?

MAPPING 2

Answer 2

• classical genetics approach

– locate (map) the gene on its chromosome by crossbreeding with individuals that carry known marker traits and collecting statistics on how frequently the mutant and marker traits are inherited together

Question 3

How do we get from a mutation to the gene?

GENOME SEQUENCING =2

Answer 3

– Sequence and compare the mutated genome to a reference genome

– Requires a good reference genome and statistical evaluation

Question 4

How do we get from a mutation to the gene?

Transposon tagging: 3

Answer 4

1 – Gene mutation caused by a transposable element

2 – Make a genomic library of the mutated organism and screen the library with a
clone for the transposable element (hybridisation).

3 – Any clone that is selected from the screening will contain the element. In the clone, sequences for the mutated gene will lie adjacent to the element.

Question 5

How do we get from a mutation to the gene? 3

Answer 5

• Mapping:

– Crossovers produce recombinant chromatids.

– The frequency of a crossover can be used to map genes on chromosomes

Question 6

Genetic linkage analysis (mapping):

Answer 6

If 1, 2, 3 and 4 are genes with known phenotypes
(genetic marker):

▪ determine the position of a mutation by determining the recombination frequencies
between the mutation and the 4 markers.

▪ In the example, the mutation has a closer link with gene 1 than with genes 2, 3 or 4.

Question 7

The position of the mutation can be determined…..

Answer 7

The position of the mutation can be determined…….

more and more accurately with an increased number
of markers in close proximity to the mutation

Question 8

NOTE: Genetic markers can be genes with known
phenotypes or Restriction Fragment Length = 3

Answer 8

Polymorphisms (RFLP), Amplified Fragment Length

Polymorphisms ((AFLP), Single Nucleotide

Polymorphisms (SNP) and many others

Question 9

Recombination Frequency

Answer 9

Recombination Frequency Figure:
Recombination frequency between two genes:

▪ Measures how much
recombination is observed in a particular experiment

➢ Recombination frequencies <
50 % ➔ the two genes are on
the same chromosome (e.g. 3 and 4) = linked

➢ Recombination frequencies =
50 % ➔ the two genes are
either far apart on the same
chromosome (e.g. 1 and 2) =
unlinked or on nonhomologues
chromosomes (e.g. 5 and 6)

Question 10

Identifying mutation in the genome using Next Generation Sequencing 2

Answer 10

1 ▪ Sequence and compare the mutated genome to a reference genome

2 ▪ Requires a good reference genome and statistical evaluation

➢ Example: Sequencing showed that lung Adenocarcinoma are heterogeneous
and can be divided into subtypes based on the genetic changes (mutations).

Question 11

Transposon tagging 5

Answer 11

1. Transposon induced mutation
2. Digest genomic DNA with a restriction enzyme that does not cut in the transposon
3. Ligate restriction fragments to form
   circular DNA
4. PCR using primers binding to the transposon
   sequence ➔ amplifies DNA from the disrupted gene that is attached to the transposon ends
5. Sequence PCR product
   to get information on the
   gene sequence that was
   disrupted by the
   transposon.

Works also if a plasmid was used to generate random mutations
➔ Method is called plasmid rescue

Question 12

Verifying that an identified mutation has caused the observed phenotype

Answer 12

1 * Many mutagenic approaches cause more than one mutation per genome.

2 * Verify that a particular mutation caused the phenotype is best done via complementation

– Transform the homozygous mutant with wt allele =
transgene complementation,
functional complementation

Question 13

o Recessive mutant: vs Dominant mutation

Answer 13

o Recessive mutant: the wt transgene should restore the wild type phenotype
= mutation caused the phenotype.

o Dominant mutation: mutant phenotype ➔ Self the transformed mutant to obtain restoration of wt
phenotype

Question 14

Verifying that an identified mutation has caused the observed phenotype

• 5

Answer 14

1. Homozygous mutation Mutant Phenotype + wt allele

2.Tranform

3. Transformed mutant
   - Recessive mutation: Wt phenotype
   - Dominant Mutation: mutant phenotype
4. Selfing
5. Offspring:
   Recessive mutation:
   25% mut, 75 % wt

Dominant mutation:
75% mut, 25% wt

Question 15

Reverse Genetics process

Answer 15

1 * Starts with a known gene or its products, mRNA or protein

2 * Disrupt function in cell via mutation or inhibitor

3 * Look for phenotype changes

4 * Assess role of normal gene product in biology

Question 16

Reverse Genetics: Three main approaches

Answer 16

1. Randomly mutate genome and find mutation in gene of interest
2. Targeted mutagenesis of gene of interest
3. Create PHENOCOPIES - effects comparable to mutations by interfering with mRNA or protein function

Question 17

Reverse genetics by random genome mutagenesis.

STEPS

Answer 17

1.Starts like forward genetics by creating RANDOM MUTATION IN THE GENOME
– Chemical, radiation or transposon mutagenesis

2 * Instead of phenotype screening localise the gene of interest (GOI)
– By MAPPING , retain only those mutants that map to the region of the GOI and perform a molecular analysis
– By PCR:
* good if mutation creates deletions ➔ PCR fragment from mutant is smaller than from wt
* If no obvious size difference: sequence and compare to wt
➔ analyse phenotype of mutations that are in the GOI

3. If transposon was used for the mutation, localise the GOI via Southern blot, RFLP or AFLP analysis ➔ look for presence of transposon in GOI (e.g. by size increase) and CHARACTERISE PHENOTYPE of GOI mutations

Question 18

advantages of Reverse genetics by random genome mutagenesis

Answer 18

• Mutagenesis is easy to do but it takes time and effort to identify mutations in the GOI
• Advantage: heritable mutations

Question 19

Reverse Genetics through targeted Gene-specific Mutagenesis = 5

Answer 19

1 * Called transgenics or targeted gene
disruption

2 * Mutate or inactivate a cloned gene
– gene knock out, small deletions, point
mutations

3 * Replace wild-type gene with altered gene by transforming the target organism

4 * Replacing occurs by a mechanism
resembling homologous recombination

5 * Labour-intensive, but once the targeted mutation is obtained, it is more straightforward to characterize.

Question 20

Reverse Genetics through targeted Gene-specific Mutagenesis

advantage vs disadvantage

Answer 20

– Problem: site directed recombination
does not work in all organisms

– Advantage: heritable mutation

Question 21

Three ways to introduce interfering RNA into cells:

Answer 21

A. inject double stranded RNA

B. Introduce a transgene with reverse repeat ➔ forms double strand after transcription

C. Introduce a transgene with two promoters in opposite direction ➔ two transcripts anneal and
form double stranded RNA

Question 22

Reverse Genetics by Phenocopying mutations using RNAi…

why and disadvantage?

Answer 22

➢ Used in reverse genetics to knock out or knock down expression of a gene

➢ Look for phenotypic changes

➢ Disadvantage: not always inherited

Question 23

Reverse Genetics by Phenocopying mutations using RNAi:

A. inject double stranded RNA - explain

Answer 23

1. dsRNA is synthesised in vitro
2. dsRNA is injected into cell.

Question 24

Reverse Genetics by Phenocopying mutations using RNAi:

explain B. Introduce a transgene with reverse repeat ➔ forms double strand after transcription

Answer 24

1. a transgene containing a reverse repeat is introduced into the genome
2. RNA transcript forms a self-complementary stem and loop

Question 25

Reverse Genetics by Phenocopying mutations using RNAi:

EXPLAIN C. Introduce a transgene with two promoters in opposite direction ➔ two transcripts anneal and
form double stranded RNA

Answer 25

1. A transgene containing 2 promoters in opposite orientations is introduced into the genome
2. complementary RNA molecules are transcribed and hybridise.

Question 26

Explain Phenocopying by Chemigenomics
or chemical genetics…

4

Answer 26

1 * Not a genetic approach

2 * Phenocopying by targeting the protein of
interest

3. Works by reducing the activity of a target gene’s protein product through
   binding of a small inhibitory molecule

4 * Can be done as forward or reverse
approach

Question 27

Phenocopying by Chemigenomics
or chemical genetics…
‘ADVANTAGE VS DISADVANTAGE

Answer 27

• Disadvantage:
  not inherited, inhibitors
  rarely 100% specific for a single protein
  ➔ observed phenotype may be due to a
  number of proteins
• Advantage:
  can be robotised
  ➔ test libraries of thousands of related small synthetic molecules for their ability to bind tightly to a specific protein.

Question 28

Phenocopying by Chemigenomics
or chemical genetics as FORWARD VS REVERSE CHEMICAL GENETICS

Answer 28

FORWARD CHEMICAL GENETICS …
1. wells with yeast colonies
2. Add one compound per well
3. Find component that produces phenotype of interest
4. Identify protein target of compound.

REVERSE CHEMICAL GENETICS
1. Protein of interest
2. Screen for compounds that bind to protein
3. Treat cells with molecules that binds to protein
4. Assay for phenotype.

Question 29

Both Forward and Reverse Genetics approaches generate

Answer 29

MUTANTS

Question 30

Analysis of Recovered mutants:

Usually large # of mutants are recovered

Answer 30

– can be ‘different hits in one gene’ or in ‘several genes in the same pathway’

• distinguish by RECOMBINATION MAPPING AND COMPLEMENTATION.

Question 31

Analysis of Recovered mutants:

Recombination Mapping: two mutations that are separable by recombination

Answer 31

➢ Example 1:

two mutations that are SEPARABLE BY RECOMBINATION must be mutations in
DIFFERENT GENES = NON-ALLELIC

Question 32

Analysis of Recovered mutants:

Recombination Mapping: MAY BE ALLELIC

• complementation test = 2

Answer 32

➢ Example 2: MAPPING IN THE SAME REGION OF THE GENOME: ‘MAY BE ALLELIC’ = represent
multiple hits in the same gene or may represent mutations in a cluster of genes

➢ Complementation test to resolve example 2

o if 2 recessive mutations complement, then in different genes

o if 2 recessive mutations do not complement, then in same gene

Question 33

Complementation test does not work with dominant mutations because

Answer 33

dominant mutations have a mutant phenotype regardless of the mutant state of the other allele

Question 34

Non-allelic, recessive mutations explain…

Answer 34

Forward genetic screens often result in more than one independently derived mutant with similar phenotypes

Question 35

QUESTION: are the mutations in the
– SAME GENE = ALLELIC MUTATIONS

– or in DIFFERENT GENES that effect the
same pathway/phenotype = NON-ALLELIC MUTATIONS

Answer 35

EXAMPLE :

A plant that lacks the
function of gene B (genotype bb) would produce mutant,

white flowers that looked just like the flowers of a plant that lacked the
function of gene A (genotype aa) ➔ non-allelic

Question 36

Complementation or cis-trans analysis:
Allelic or Non-allelic mutations

Answer 36

1 * In a typical complementation test, the genotypes of two parents are unknown

• Case 1: F1 progeny all have a mutant phenotype, no complementation ➔ mutations are allelic, written as m1/m2 (here aa)
• Case 2: F1 progeny are all wild-type, complementation ➔ mutations are non-allelic, written
  as: m1 +/ + m2 or + m1 / + m2 (here AaBb) = two genes

Question 37

Complementation group:

Answer 37

— a group of mutations that fail to complement one another ➔ all allelic

• NOTE: complementation test only works with recessive mutations, not with dominant mutations

Question 38

Distinguishing Loss of Function from Gain of Function:

RECESSIVE VS DOMINANT MUTATIONS: 4

Answer 38

• Important information about function can be gained by knowing what has gone wrong in a mutant

1. • RECESSIVE MUTATIONS :
     are usually loss of function alleles of a

HAPLOSUFFICIENT gene:
– one dose is enough ie. A/A = Aa, same phenotype

2 * DOMINANT mutations are more interesting and more ——–

INFORMATIVE: gene dose can make a difference:
– example: A/A > A/a, different phenotype

3. • Special tests are needed for dominant mutations to distinguish ——Loss of Function
     from Gain of Function dominant mutations

Question 39

Dominant amorphic null mutations
– complete loss of function

Answer 39

A dominant null mutation of a haploinsuffcient gene with a single copy of the wild-type allele does not make enough gene product to generate a wild-type phenotype.

Identify such mutants by comparing:

Question 40

Amorphic allele:

Answer 40

The gene is required in two copies to elicit a normal phenotype.

Question 41

Dominant hypomorphic, leaky mutations:

Answer 41

A dominant hypomorphic mutation of a haploinsuffcient gene —-with a single copy of the
wild-type allele does not make enough gene product to generate a wild-type phenotype but the phenotype is less severe than in a deletion mutant.

This is caused by the mutated gene still making some gene product.

Question 42

Dominant Gain of Function mutants

1) Hypermorphic mutation

2

Answer 42

1 * A hypermorphic mutant makes more gene product per gene dose than the wt but the gene product itself is normal

2 * The mutation leads to a novel phenotype that is less severe in a deletion mutant and more severe
In a duplication of the wt allele mutant.

Question 43

Dominant Gain of Function mutants

2) Neomorphic mutations

Answer 43

1 * A neomorphic mutant produces a novel gene activity not characteristic of the wt.

– Example:
a neomorphic mutation may have fused the coding regions of two genes. The new
gene product did not exist in the wt.
– Another example:
a neomorphic mutation takes place in a gene promoter which changes the expression pattern of the gene. The gene product will now be expressed in tissues where it is not expressed in the wt.

2. • These mutations produce novel, unpredictable phenotypes

Question 44

1. What is the purpose of mutational dissection

Answer 44

FUNCTION

Question 45

What are the general strategies

Answer 45

• Forward & Reverse

Question 46

How are mutations characterised

Answer 46

• Selection & Screening

Question 47

Ways to create mutations

Answer 47

– EMS, ENU, X rays, spontaneous, transposons etc

Question 47

How can we verify the cause of the phenotype

Answer 47

– complementation

Question 48

How can we isolate the mutated gene

Answer 48

– mapping, genome sequencing, transposon
tagging