Lecture 14

Question 1

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

Answer 1

Classical genetics” strategy – starts with phenotype
• A series of mutants with defects in a particular phenotype may be linked in the same molecular pathways/functions
• Enables the wild-type genes for this pathway to be identified and studied

Question 2

Step 1:

Answer 2

choose a good organism, mutagenize, screen for mutants with the desired phenotype eg temperature sensitive mutants – grows at low temp but doesn’t at high temp or genetic analysis of cell cycle. Looked for mutants that could not divide

Question 3

Describe the yeast experiment

Answer 3

• Yeast grows in a length ways and then divides to produce 2 symmetrical cells
• Behaved normally at a low temperature
• High temperature wouldn’t divide so formed elongated cells
• Looked for elongated cells – cdc,

Question 4

Step 2

Answer 4

: define genes responsible for these phenotypes: genetic complementation tests
• Cdc1, cdc2 etc. genes
• Looked at where they progressed through the cell cycle at different temperatures – some genes were responsible for S, G1 or M phase

Question 5

Step 3

Answer 5

Cloning genes: by mutant complementation
• Take a gene library from organism wild type cells and transform mutants from the library
• Isolate transformants which rescue or restore a wildtype phenotype which survive at high temperatures
• Recover plasmids
• Cloned gene is collected

Question 6

Step 4

Answer 6

• Compare predicted protein sequence with database sequences
• BLAST – Basic Local Alignment Search Tool
• Gives clues as to functions
• Put genes back into mutant cell
• Example: cdc 17 – DNA ligase, Nda3 – b-tubulin, cdc 2 – protein kinase

Question 7

Describe mutant rescue

Answer 7

• Looking for functional homologues but used a human gene library
• Used cDNA library which is a copy of RNA
• Cdc2 – like proteins discovered in all eukaryotes
• Cyclin dependent kinases
• Control the cell cycle
o The eukaryotic cell cycle is principally controlled by protein phosphorylation.
o The CDKs responsible for cell cycle control were discovered by genetic analysis of the cell cycle.
o The regulatory subunits of these protein kinases were discovered by biochemical and cell biological studies.

Question 8

Describe sequence alignments

Answer 8

similiary infers homology – sequences that are related by evolutionary descent. Divergence between two homologous sequences is proportional to the mutation/recombination rate

Question 9

Describe what happens if two sequences are homologous

Answer 9

they started from one sequence which duplicated to give two genes similar – one which picked up a mutation (paralogous genes in same organisms) or speciation (orthologous genes in different organisms)

Question 10

Describe evolutionary trees

Answer 10

Carl Woose – Bacteria, Archaea and Eukaryota – looked at ribosomal sequences – phylogenetic tree