Yeast Lecture 2 Flashcards
What is forward genetics?
Select mutants and characterize the genes
How is dominance/recessivity determined in yeast?
By backcrossing into the wild type
What is a allelic mutation?
A mutation on the same gene
What is reverse genetics?
After findings from biochemical analyses, „Proteomics“ or „Genomics“, the analysis of the physiological relevance of a gene’s function
Gene deletion via homologouse recombination strategy
A Marker C
A B C
By cloning into site between A and C = B is deleted
What genes are usually used as selectable markers?
Resistance genes
How do you test for your integrated Gene in case of gene deletion?
One primer inside of marker and one outside
What is introduction of Tags via plasmid integration?
Integrative plasmid is linearalized via restirction to stimulate HR
A B C
A B C tag Marker C
leads to gene duplication
How do you test for your integrated Gene in case of Tag introduction?
One primer in Tag one outside of integrated plasmid
What is the short flanking homology regions strategy
Using short homolgy arms as part of the PCR primers to direct the cassette to target sequence
What are different types of plasmids
Integrative plasmid (contain selectable markers)
Centromeric plasmid (1 copy per cell)
high copy plasmid (15-100 copys per cell)
Types of suppressor mutations/genes
intragenic Revertants
extragenic Pseudorevertants
informational (e.g. suppressor tRNAs)
metabolic
What are Metabolic extragenic suppressors often used for?
To identify new genes in a pathway
What is a bypass suppressor?
Suppressor mutation that indirectly affects the interacting proteins and they are gene specific
If they directly affect the interacting proteins they are usually allele specific
How can site-specific mutations be stably introduced ?
2 Step gene transplacement
Plasmid integration -> plasmid popout
When is a mutation epistatic?
If the phenotype caused by one mutation is not enhanced by another mutation in the same pathway, the former is epistatic to the latter
When is a mutation synthetic?
If phenotype of mutation is enhanced by another mutation
What is the Yeast two hybrid System?
Detects Protein-Protein Interactions
DNA Binding Domain (BD) and the transription-Activation Domain (AD)
of transcription factor Gal4 is connected via a linker domain
If bait and Prey are in close proximity -> gene is transcribed
