Yeast Lecture 2

Question 1

What is forward genetics?

Answer 1

Select mutants and characterize the genes

Question 2

How is dominance/recessivity determined in yeast?

Answer 2

By backcrossing into the wild type

Question 3

What is a allelic mutation?

Answer 3

A mutation on the same gene

Question 4

What is reverse genetics?

Answer 4

After findings from biochemical analyses, „Proteomics“ or „Genomics“, the analysis of the physiological relevance of a gene’s function

Question 5

Gene deletion via homologouse recombination strategy

Answer 5

A Marker C
A B C
By cloning into site between A and C = B is deleted

Question 6

What genes are usually used as selectable markers?

Answer 6

Resistance genes

Question 7

How do you test for your integrated Gene in case of gene deletion?

Answer 7

One primer inside of marker and one outside

Question 8

What is introduction of Tags via plasmid integration?

Answer 8

Integrative plasmid is linearalized via restirction to stimulate HR
A B C
A B C tag Marker C
leads to gene duplication

Question 9

How do you test for your integrated Gene in case of Tag introduction?

Answer 9

One primer in Tag one outside of integrated plasmid

Question 10

What is the short flanking homology regions strategy

Answer 10

Using short homolgy arms as part of the PCR primers to direct the cassette to target sequence

Question 11

What are different types of plasmids

Answer 11

Integrative plasmid (contain selectable markers)
Centromeric plasmid (1 copy per cell)
high copy plasmid (15-100 copys per cell)

Question 12

Types of suppressor mutations/genes

Answer 12

intragenic Revertants
extragenic Pseudorevertants
informational (e.g. suppressor tRNAs)
metabolic

Question 13

What are Metabolic extragenic suppressors often used for?

Answer 13

To identify new genes in a pathway

Question 14

What is a bypass suppressor?

Answer 14

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

Question 15

How can site-specific mutations be stably introduced ?

Answer 15

2 Step gene transplacement
Plasmid integration -> plasmid popout

Question 16

When is a mutation epistatic?

Answer 16

If the phenotype caused by one mutation is not enhanced by another mutation in the same pathway, the former is epistatic to the latter

Question 17

When is a mutation synthetic?

Answer 17

If phenotype of mutation is enhanced by another mutation

Question 18

What is the Yeast two hybrid System?

Answer 18

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