Dissection of Gene Function part I: Forward Genetics

Question 1

We have two principal approaches to learn about gene function:

Answer 1

Forward and Reverse Genetics

Question 2

Two strategies which are based on inheritable changes:

LIST AND DEFINE BOTH

Answer 2

1. Forward Genetics - Look for mutant phenotypes of interest followed by molecular analysis of mutants
2. Reverse Genetics
   – Mutate a known
   gene - infer function by change in phenotype compared to wt

Question 3

phenocopying

Answer 3

an environmental induced, non-heriditary phenotype of one individual which is identical to the genotype-determined phenotype of another individual.

Phenocopying causes
disruptions that are (usually) not inherited.

Question 4

Gene function and study of Genetics

Answer 4

Genetics enables us to study abnormal gene function in mutants to inform us about normal gene function.

Question 5

Forward Genetics can be done in two principal ways:

Answer 5

• Forward Genetics analyses heritable phenotype of a mutant at the genetic level then perform a molecular analysis of the mutant.

1 * Scenario 1: Survey the genome for ALL genes that contribute to a particular biological process
– Start with wild type genome
– Mutagenize randomly a large population of wt genomes
– Systematic survey for mutations that share the desired phenotype suggesting that these mutation affects the process under investigation.

– Perform molecular analysis of all these mutants

• Scenario 2: Mutated gene causing a phenotype is known. More mutations in that gene should be investigated to better understand that single gene’s function

– Collection of mutants is tested for mutations in that particular gene followed by
cloning and sequencing

– Correlate mutations with observed phenotypic changes (e.g. severity)

Question 6

Forward Genetics:

Scenario 1: Survey the genome for ALL genes that contribute to a particular biological process = 4

Answer 6

– Start with wild type genome

– Mutagenize randomly a large population of wt genomes

– Systematic survey for mutations that share the desired phenotype suggesting that these mutation affects the process under investigation.

– Perform molecular analysis of all these mutants

Question 7

Scenario 2: Mutated gene causing a phenotype is known.

More mutations in that gene should be investigated to better understand that single gene’s function =2

Answer 7

– Collection of mutants is tested for mutations in that particular gene followed by
cloning and sequencing

– Correlate mutations with observed phenotypic changes (e.g. severity)

Question 8

Choice of mutagens: 2

Answer 8

• Either use naturally occurring mutants
• Or find a mutagen that induces mutations

– Transposons
* e.g. maize Ac/Ds transposons
– Transform one plant with an Ac element, another with a Ds element

» Crossing of the two transgenic plants activates Ds which transposes and interrupts genes

» Outcrossing Ac removes transposase ➔ Ds element trapped and
tags the mutation ➔ easy to isolate the mutated gene

– Chemical mutagenesis:

• e.g. using EMS (Ethyl methanesulfonate), MMS (Methyl methanesulfonate),
  ENU (Ethyl-nitrosourea) ➔ requires uptake of mutagen into the cell

– Radiation mutagenesis

• UV: good for microbes
• X rays, gamma rays: cause large-scale changes such as intragenic deletions

Question 9

Choice of mutagens: Transposons

Answer 9

Transposons
* e.g. maize Ac/Ds transposons

– Transform one plant with an Ac element, another with a Ds element

» Crossing of the two transgenic plants activates Ds which transposes
and interrupts genes

» Outcrossing Ac removes transposase ➔ Ds element trapped and tags the mutation ➔ easy to isolate the mutated gene

Question 10

Choice of mutagens: Chemical mutagenesis

Answer 10

Chemical mutagenesis:

• e.g. using EMS (Ethyl methanesulfonate), MMS (Methyl methanesulfonate),
  ENU (Ethyl-nitrosourea) ➔ requires uptake of mutagen into the cell

Question 11

Choice of mutagens: Radiation mutagenesis.

Answer 11

– Radiation mutagenesis

• UV: good for microbes
• X rays, gamma rays: cause large-scale changes such as intragenic deletions

Question 12

Chemical mutagenesis using EMS

Answer 12

1* mutagenic, teratogenic, and possibly carcinogenic

2 * Alkylates guanine

3 * During replication, alkylated guanine will base pair with T instead of C

4 * After another round of replication, the base pair
will change to a T-A base pair

➔ transitional point mutation from C-G to T-A

Question 13

Ionizing or non-ionizing radiation mutagenesis

Define and Explain them:

Answer 13

• IONIZING RADIATION was the first mutagen that efficiently and reproducibly induced
  mutations in a multicellular organism.

– X rays, gamma rays (γ), beta particle radiation (β), and alpha particle (α)
radiation

– X-rays and gamma rays cause single strand breaks

• NON-IONIZING RADIATION
  – UV radiation, like that in sunlight, is non-ionizing.

Causes thymine dimers. If not repaired lead to errors during replication

Question 14

Mutation rate versus Mutation dose:

EXPLAIN Mutation rate: + 3

Answer 14

• Mutation rate: frequency of new mutations in a single gene or organism.

– Ensure that:
* mutagen produces sufficient mutations to enable recovery of desired
mutations

• mutagen does not produce more than one mutation per genome ➔ multiple mutations make genetic analysis difficult or kill the cell

– Usually: a mutagen dose that leads to 50% survival rate is aimed for.

Question 15

Mutation rate versus Mutation dose:

DEFINE + WHY DIFFICULT OT ACHIEVE 3

Answer 15

Ideally try for SATURATION MUTAGENESIS:
all genes in the genome that confer a specific phenotype are mutated.

Difficult to achieve because:
1 - Gene size determines probability of mutation in that gene

2 - Mutations are often pleiotropic ➔ they have numerous effects on phenotype

3 - Mutations can be severe: death, infertility

Question 16

Mutations are rare:

Answer 16

frequency ≤ 10-5

➔ Effective assay systems needed to recover desired mutants

Question 17

Assay Systems for Forward Genetics:

Genetic Selection 4

Answer 17

1 – Killing or inhibiting wild types and non desired mutants, mutants survive

2 – Easy to do

3 – Advantage: only desired mutant survives

4 – Often used for microbes – supplement growth medium with a selective agent

Question 18

Selective agents may be: 5

Answer 18

1 * Specific nutrient toxic for non-mutant

2 * Growth on absence of a specific nutrient

3 * Inhibitors

4 * Pathogens

5 * Antibiotics

Question 19

genetic selection vs genetic screening

Answer 19

Mutagen into Bacteria growing in liquid culture.

to

1. Genetic Selection:
   Individuals lacking phenotype of interests are killed.
   - Individual with mutant phenotype of interest survives

or
2. Genetic Screen
- Numerous individuals survive
- phenotype of each survivor must be examined
—individual with mutant phenotype of interest is found.

Question 20

Prototroph: 2

Answer 20

1 – Organism or cell capable of synthesizing all its metabolites from inorganic material, requiring no organic nutrients

2 – Can grow on minimal
medium

Question 21

Autotroph: 2

Answer 21

– Can synthesize its food from inorganic substances

– Can grow on minimal medium but requires heat or light as energy source

Question 22

Example of a Forward Genetic Selections
– selection of auxotroph mutants

Auxotroph:

Answer 22

– Mutant organism, that requires the addition of a particular organic compound they cannot synthesize

– Do not grow in Minimal medium, require Opposite of autotroph – self-feeding

Question 23

Example of a Forward Genetic Selections

Answer 23

Selection of auxotroph mutants in filamentous Fungi using filtration enrichment.

1 * Prototrophic wt filamentous fungi cells form ‘fuzzy balls’ when grown on liquid minimal medium

2 * Auxotrophic mutant cells do not grow

3 * Wild type is filtered off and discarded

4 * Mutant cells are in the filtrate and can be grown on minimal medium supplemented with the required organic compound
– e.g if leucin is the requirement compound then leucin auxotrophs will grow

5 * Can do this with a supplementation of a variety of organic compounds to select for different auxotrophies
* ➔ investigate metabolic pathways

Question 24

Example of a Forward Genetic Selections - simplified 4

Answer 24

1. Minimial medium:
   - Prototrophic wild types
   - nutrional mutants (auxotrophs)
2. Prototrophs held by filter
3. Auxotrophs pass through
4. Onto plate
   Auxotrophs can grow…Leucine-containing medium

Question 25

Testing Neurospora crassa strains for auxotrophy using forward genetic selection

Answer 25

1. N. crassa: model organism, a filamentous fungi, type of red bread
   mold

2.* 20 progeny from a cross adleu+ x ad+
leu–inoculated on minimal medium (Min) or
minimal medium with adenine (Ad) or leucin (Leu) or both

3 * ad mutants cannot produce adenine, leu mutants cannot produce leucine

4 * Look for colonies growing on the different media to determine which auxotropy they have and to isolate a double auxotroph.:

Question 26

Forward Genetic Selections can also be done to identify mutants in animal behaviour

PHOTOTAXIS MUTANT

Answer 26

Wild type Drosophila shows

PHOTOTAXIS: orientates itself and flies towards light in the T-maze

PHOTOTAXIS MUTANT screen identifies Drosophila mutants that lost the ability to fly towards light ➔ flies appear at the light and dark side of the Tmaze with equal probability

Question 27

Assay Systems for Forward Genetics:

Genetic Screens what is it?
disadvantage/advanatge

Answer 27

Genetic Screen
– Identify desired phenotype

– More work

– Advantage: a greater range of phenotypes
can be looked for

Question 28

Genetic screens can be used to dissect any biological process = 4

Answer 28

1 – Development

2 – cell cycle

3 – signal transduction

4 – metabolism etc

Question 29

Genomics has facilitated Forward Genetic screening

Answer 29

• application of high-throughput approaches to
  Genetics

Question 30

Morphogenesis:

Answer 30

• Morphogenesis: development of form

Question 31

Mutagenize large population of haploid cells and plate those

Answer 31

– Haploid ➔ even recessive mutations can be observed, cannot be covered by a dominant wt allele

Question 32

Forward Genetic Screens to identify Morphogenesis mutants in Neurospora = 3

Answer 32

1* Mutagenize large population of haploid cells
and plate those
– Haploid ➔ even recessive mutations can be observed, cannot be covered by a dominant wt allele

2 * Identify changes in colony morphology

3 * ➔ hundreds of loci identified that are involved in tip growth and branching of the filamentous fungus.
– mutations found in many cytoskeletal genes such as actin, dynactin and dynein

Question 33

yeast

Answer 33

single cell eukaryotic fungi

Question 34

• Saccharomyces cerevisiae – budding yeast (beer, bread) =2

Answer 34

– Outgrowth from the parent produces a bud

– first complete DNA sequence of a eukaryotic
genome (1996)

Question 35

Schizosaccharomyces pombe – fission yeast

Answer 35

– Fission: results in two identical individuals by
splitting

Question 36

TAPOYG - The Awesome Power Of Yeast Genetics:

Both are model eukaryotic organisms for genetics and molecular biology because

Answer 36

– Many genes in yeast and mammals encode
very similar proteins

– Investigating yeast mutants has many valuable outcomes for research in other eukaryotic organisms, including humans

Question 37

Conditional heat sensitive
mutants

Answer 37

1 * Temperature-sensitive mutants are important tools for understanding the role of
essential gene(s).

2 * Often, they result from amino acid changes due to mutations of genes.

3 * Restrictive temperature: high growth temperature at which the mutation causes the protein to malfunction or not function at all ➔ often deleterious ➔ mutant phenotype

4 * Permissive temperature: lower temperatures at which the mutation does not impact on protein function ➔ wt phenotype

Question 38

What happens in Permissive Temperatures? 5

Answer 38

1. Unfolded polypeptide
2. Folding
3. Binding to cognate partner
4. Active protein
5. WT phenotype

Question 39

What happens in Restrictive temperatures?

Answer 39

Unfolded polypeptide

1. Unfolding or misfolding
2. proteolysis or Aggregation
3. Aggregation -> Decreased amount of active protein
4. TS phenotype

OR
1. folded and stable
2. Low affinity for partner
3. TS phenotype

Question 40

Visual identification of mutants with abnormal celldivision cycle - cdc mutants

Answer 40

1 – Expectation: many cdc mutants are lethal

2– Screen for conditional heat sensitive mutants:
* Nobel prize for Leland Hartwell and Paul Nurse

Question 41

Mutations block the mitotic cell cycle at different,
specific points 4

Answer 41

(a) Abnormal mitosis - DNA segregates irregularly along spindle

(b) haploids enter meiosis

(c) mutants elongate without dividing

(d) Mutants arrest without budding

Question 42

Forward Genetic Screens to dissect the Cell Cycle in Yeast using heat sensitive screens

What identified? Comparative genomics?

Answer 42

• Identified many proteins that regulate and control the
  cell cycle progression
• Comparative genomics has shown that these same genes are at work in the cell cycle of humans, and that
  many of these genes are defective in cancers.

Question 43

A Visual Forward Genetic Screen for nuclear division
defects in Aspergillus

Answer 43

• A visual screen for altered nuclear division
  (screened at lower than normal growth temperature, see yeast)
• 3 classes of heat sensitive mutants showed key players in cell division and growth:

– nimA - never in mitosis
* codes for a kinase

– bimC - blocked in mitosis
* codes for kinesin, a motor protein that moves organelles on cytoskeleton

– nudA - nuclear distribution
* a dynein subunit, also moves organelle

Question 44

Aspergillus

Answer 44

is a filamentous fungus, genetic
model organism

Question 45

A Visual Forward Genetic Screen for Dissection of Development in Zebrafish

Answer 45

• Small, develop rapidly, and produce many offspring ➔ good model for vertebrate development
• Transparent embryos ➔ easy to observe abnormalities in early development
• Male take up a chemical mutagen ENU (Ethyl-nitrosourea)* via tank water
  – mutagenised males produce BASE-SUBSTITUTED sperm with many different mutations
• *****F1 contains one normal chromosome (mother) and one mutagenised (father)
  – F1 males x WT females
• Offspring interbreed to reveal recessive phenotypes

Question 46

Forward Genetic Screens made easier
using haploid Zebrafish =4

Answer 46

1 * Speed up zebrafish mutant screen by producing haploid offspring

2 * Achieved by UV irradiation of F1 males
– destroys sperm nuclei (heavily
mutagenized) so they cannot
contribute sperm genomes to the zygote
but can still fertilize egg

3 * Offspring don’t develop to adults but can look for mutants in early development

4 * Half of the offspring carries newly induced mutation inherited from the
female parent

Question 47

Forward Genetic Screen to Identify Enhancer elements in eukaryotic genomes using enhancer trapping

Answer 47

1 * Eukaryotic enhancers (DNA regulatory sequences) act as enhancers of transcription of genes whose transcription start site is nearby

2 * Hunting for enhancer sequences using enhancer trap: randomly insert a transgenic reporter construct (e.g. GFP) into genome ➔ reporter is activated if it inserts near an
enhancer.

3 * Expression of reporter gene due to enhancer activity can be observed

4 * Identify enhancer elements that drive particular gene expression pattern, e.g. developmental, organ specific…

5 * If the enhancer trap is on a transposon ➔ can be mobilised and move to other
genome positions to identify further enhancers

Question 48

Minimal Promoter - Reporter Gene =

Answer 48

Minimal Promoter - Reporter Gene = No or weak expression

Enhancer + Minimal Promoter to Reporter gene = strong GENE EXPRESSION

Question 49

Forward Genetic Screen to Identify Enhancer elements
involved in Drosophila = 2

Answer 49

• Genes A and B in the Drosophila genome are regulated by enhancer elements in a developmental
  fashion.
• Insertion of an enhancer trap leads to activation of a
  reporter gene by the enhancers. The activation is
  development specific