E.coli Lecture 4

Question 1

What is the Toxin/Antitoxin System

Answer 1

Normally Toxin and Antitoxin Dimerize and neutralize each other.
Upon Phage infection Antitoxin is degraded and cell dies due to toxin.

Question 2

How do you discover new defense Systems in prokaryotes

Answer 2

You search the genome for known defense systems and co-encoded unknown genes -> candidate defense system
-> check functionality of candidate defense systems in “model” organisms

Question 3

What are some Bacterial defense system features

Answer 3

Defense Systems mainly encoded by variable gene pool
Defense Systems are clustered

Question 4

Why does Saccharomyces cerevisiae have so many more genes than S.pombe?

Answer 4

Due to a genome duplication event S. cerevisiae gained a lot of genes

Question 5

What is S. cerevisiae metabolism under glucose?

Answer 5

Under sufficient Glucose its metabolism is Glycolyses and alcoholic fermentation
-> Releases CO2 and Ethanol

Question 6

What hinders S. cerevisiae from using respiration or other carbon souces under sufficient glucose?

Answer 6

Catabolite repression

Question 7

What are the growth phases of S. cerevisiae?

Answer 7

Lag phase -> Exponential phase -> Stationary phase

Question 8

What is the diauxic shift?

Answer 8

The moment S. cerevisiae stops using glucose and starts respiration with its ethanol

Question 9

What is S. cerevisiae special feature during cell division?

Answer 9

Closed mitosis -> Nuclear membrane remains intact

Question 10

What are heterothallic strains ?

Answer 10

Can be kept in the haploid state as two different mating types. Upon combination (conjugation) they will form a diploid zygote which can reproduce.

Question 11

What does nitrogen + carbon starvation do in diploid yeast cells?

Answer 11

They undergo sporulation -> yields 4 different haploid spores.

Question 12

How can S. cerevisiae switch its mating type?

Answer 12

By gene conversion. Inducing a double strand break via the HO endonuclease

Question 13

Homo vs Heterothallic strains

Answer 13

Homothallic strains have a functional HO endonuclease -> very frequent mating type switch -> nearly all cells end up as diploid

Question 14

Whats a crossing of a Parental Ditype

Answer 14

2 AB -> A - B
-> A - B
2 ab -> a - b
-> a - b
no crossover

Question 15

Whats a crossing of a non-Parental Ditype

Answer 15

2 Ab -> A - B
-> A - B
2 aB -> a - b
-> a - b
4 strand double crossover

Question 16

Whats a crossing of a Tetratype

Answer 16

AB -> A - B
ab -> A - B
Ab -> a - b
aB -> a - b
single corssover

Question 17

What are the different cell types of S.cerevisiae?

Answer 17

Mat a or Mat alpha

Question 18

How can S. cerevisiae switch its mating type?

Answer 18

By gene conversion. Via conversion casette
The cells carry the active MAT locus and two silent ones with both a and alpha
HO endonuclease can introduce a double strand break and switch one casette for the other.

Question 19

How is the mating type switch controlled in S. cerevisiae?

Answer 19

By HO endonuclease initiation and by pheromones. a and alpha factor

Question 20

What is the nomenclature in S. cerevisiae?

Answer 20

GOI1=wild type/dominant allel
goi1-1 = recessive mutant with allel number
goi1-S313T = recessive mutant allele with known mutation
Goi1 or Goi1p protein

Question 21

What is the Psi-prion?

Answer 21

Impairs STOP codon recognition
Leads to ade1 expression and white colonies.( can grow on medium lacking adenine)
If ade1 is not present = red colonies

Question 22

Why are restirciton sites not cut by RMS in the host?

Answer 22

Because they are methylated

Question 23

What is the pAgo system?

Answer 23

pAgo only cuts multicopy nucleic acids (prevents host DNA from being cut)
uses cut DNA as guide to be more specific

Question 24

What happens if ade1 is not present in yeast on YPD culture medium?

Answer 24

Red colonies

Question 25

How do you use error prone PCR to generate conditional allels in a gene?

Answer 25

1. Generate strain with conditionally expressed GOI
2. Amplify GOI by error prone PCR
3. Introduce PCR products and linearized plasmid
   into yeast cells (⇢in vivo recombination)
4. Select viable transformants on glucose media
5. Test transformants for inability to grow at 37°C

Question 26

How are the yeast colonies selected in the two step gene replacment?

Answer 26

By FOA which is toxic in the presence of Ura.
Plasmid contains Ura but is lost over time. By adding FOA cells are killed who still have the plasmid.

Question 27

How does Yeast two hybrid work?

Answer 27

Gal-BD
Gal-AD
Linker
Linker is deleted and GalBD and GalAD cannot interact -> gene is not transcribed
Only when Bait and Prey are in close proximity -> gene is transcribed
Selection on medium lacking gene of intrest.