Forward Genetic Strategies

Question 1

What are forward genetic approaches?

Answer 1

seek to find the genes encoded by DNA that are responsible for a phenotype of interest

starts with phenotype —> genotype
have to have a biological question, to generate mutants, identify them and unpick their pathways

Question 2

What are the 4 genetic steps?

Answer 2

1. Isolate mutants with phenotype of interest
2. Define genes responsible for the phenotypes
3. Clone or identify the genes - isolate sequence
4. Analyse the genes to predict encoded proteins

Question 3

What factors influence choice of organism?

Answer 3

biological process
fast reproductive time
easy to handle in the lab

Question 4

Example organisms chosen

Answer 4

unicellular eukaryote= yeast e.g. s.pombe
zebra fish - can see different structures and grow quickly

picking right model critical as want to be able to mutate it and see the effect

Question 5

Examples of physical mutagens

Answer 5

UV
Ionising radiation (x-rays, gamma rays)

Question 6

Examples of chemical mutagens

Answer 6

Base analogues
Alkylating agents

Question 7

Examples of biological mutagens

Answer 7

Transposable elements
Random gene integration

Question 8

How do you screen for the desired phenotype?

Answer 8

survive in one condition, lethal in another
stamp replica plates
- put them at different temperatures
- some fail to form a colony, lethal point mutations now taking their effect

cdc mutants: elongated cells at higher temperature (mutant)

Question 9

How do you then define the genes responsible for the phenotype?

Answer 9

genetic complementation tests
determine if from same or different genes
take 2 mutant strains and cross together

cdc= mutant on same gene
selected only cells that has single gene mutations
could copy genomes but wouldn’t separate properly

Question 10

How do you clone and identify the genes?

Answer 10

Take mutant gene
make a gene copy from cDNA library
in ORF (open reading frame)

All bacteria that grow will have short pieces of s pome genome
library of plasmids with short pome genome
cells must have been rescued by plasmids
then identify what part of genome has recovered it

Question 11

What are the cloning steps?

Answer 11

1. conditional lethal cdc mutant + cdc wild type library in vector
2. transform, then select
3. cells that survive at 36oC
4. recover plasmid in e.coli
5. cloned gene

Question 12

How do you analyse the genes to predict encoded proteins?

Answer 12

Compare predicted protein sequence with database sequences
If sequence looks like BLAST sequence, likely to have same function as already identified

Question 13

What is mutant rescue?

Answer 13

cdc2-like proteins discovered in all eukaryotes
now called CDKs - control cell cycles

protein so well conserved if you take human sequence it will grow and rescue a year cell

Question 14

What is S.pombe Cdc2 kinase

Answer 14

encodes a protein kinase implicated at G1-S and G2-M control points in cell cycle

homologues of cdc2 exist in all other eukaryotes
control the cell cycle

Question 15

how is the cell cycle controlled?

Answer 15

by protein phosphorylation
cyclins of the protein kinases discovered by biochemical & cell biological studies