DNA Repair/Slecting Mutants/Gene Transfer

Question 1

DNa Repair

Answer 1

SOS Repair
Light Repair
Dark Repair

Question 2

SOS Repair

Answer 2

add bases even if they are mispaired, may be still a mutant

Question 3

Light Repiar

Answer 3

break T-T dimer, using photoactivation to break the dimer, to cut out mismatched DNA. T-T+Photolyase+light=T T
how?
A.endonuclease-cuts of damages area
B.results ssdna serves for template for dnapol
c.Dna ligase links new piece to rthe rest

Question 4

Why select for mutants?

Answer 4

to find a bacerial mutation that is beneficial

Question 5

How do you select for mutants?

Answer 5

Direct screening and indirect selection

Question 6

Direct screening

Answer 6

plate pssible mutants you hope can survive

Question 7

goals of creating mutants

Answer 7

mutant survives where prototroph cannot or vice versa

Question 8

Example of direct screening/selection

Answer 8

1. Mutate (using radiation, chemcial, base analog)
2. Pt microbes on media containing substance causing death
3. look for growth, only mutants should grow

Question 9

Indirect Selection

Answer 9

obtain an auxotroph which has increased requirements due to mutation

Question 10

Example of indirect selection

Answer 10

protorpoh-mutate-auxotroph (auxotroph will not grow on basic media needs enrichment)

HIS+->mutat->HIS-
His+ has gene to make histidine H- doesnt

Question 11

How is HIS- found?

Answer 11

replecating plate is required, plate with histidine the stamped with and without histidine using velvet template
practical discovery=AMES TEST

Question 12

AMES TEST

Answer 12

look for increase in reversion rate of mutant hus as HIS-

Question 13

Gene transfer

Answer 13

alter genetic information by moving genetic info from cell to cell

Question 14

genome

Answer 14

all nucleotides sequence

Question 15

genotype

Answer 15

all posible genes for use

Question 16

phenotype

Answer 16

appearance

Question 17

If dna is changed at nucleotide level what occurs

Answer 17

genome changes
genotype may change
phenotype may change

Question 18

Three methods of gene transfer

Answer 18

1. Transformation
2. Transduction
3. Conjugation

Question 19

1. Transformation

Answer 19

naked dna transfer directly to a recipient (griffith experiment)

Question 20

2.Transduction

Answer 20

dna transfer using vector (virus, plasmid)

Question 21

3. Conjugation

Answer 21

dna transfer resulting from CONTACT between cells

SEX PILUS

Question 22

Gene Transfer features

Answer 22

only part of dna is trasnferred,
processes:
a.nuclease cleaves recipient dna and same with donors (using endonuclease/restriction)
b.donor dna is jioned with ligase
c.exonuclease chews up/any excised dna
transfer of dna 1/100
d. use selection techniques to find recombinants (probes)`

Question 23

Transformation ideal conditions

Answer 23

1. cell must be competent=ability to take dna
2. not all cells are competent
   can use electroporation and CaCl2 to help

Question 24

Transduction ideal conditions

Answer 24

1. Virus (BACTERIOPHAGE) injects DNA into bacterial cell
2. piece of dna is packaged in virus
3. new phage infects new cell
4. donor dna integrates with host dna
5. recombination of dna has occured

Question 25

Conjugation ideal conditions

Answer 25

“bacterial mating” dna is transfered in one direction
donor has a special plasmid which allows trasnfer (F PLASMID)
F Plasmid-carries information which has genetic info for sex pilus and genetic info for toxins
Sex Pilus-attachets to recipient cell anda llows passage of part or all F Plasmid in form of SSDNA
F+ has plasmid code
F- Doesnt

Question 26

R factors

Answer 26

antibiotic resistant genes carried with plasmid
plasmids can replicate autonomously
plasmids can have >1 copy per cell

Question 27

How plasmid genes change work in conjuction

Answer 27

with TRANSPOONS

transposistion fo vancomyocin resistant gene from VRE to a plasmid within MRSA creation of VRSA