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Flashcards in Bacterial Genetics 1 Deck (54):
1

The best way to see what a gene does

- break it and see what happens

2

the best way to tell what gene controls a process

break lots of genes and look for an organism that can't do the process

3

mutation

change in DNA sequence

4

isogenic

all genes except one are the same

5

species

a population of microorganisms with similar characteristics

6

clone

a population of cells that are genetically identical

7

genotype

the specific set of genes present in a cell

8

phenotype

the collection of characteristics that are available

9

wild type strain

- a recognized "type" strain, which is a clonal population of a particular species
- has the identical genotype and phenotype

10

mutagens

- chemicals
- ionizing radiation
- UV radiation
- agents that increase mistakes in DNA replication

11

point mutation

- change of one base to another

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point mutation outside of protein coding region

- no effect

13

silent mutation

codon still encodes same AA

14

missense mutation

codon codes for different AA

15

read through mutation

- normal termination sequence is mutated and no longer stops translation

16

nonsense mutation

change to termination codon.

17

frameshift mutation

- if one of two bases added or deleted within the coding region
- new AA sequence downstream

18

spontaneous mutations

- 1 mutation/generation
- 10^-6 to 10^-8 per generation
- errors not fixed in DNA replication
- DNA damage

19

UV induced damage

- wavelength less than 280 nm
- form pyrimidine dimers
- DNA pol will stall or skip dimer completely or misincorporate other nucleotides
- UVA at shorter wavelengths can break backbone

20

ionizing radiation

- radiation forms chemical free radical, which can cause double strand breaks in DNA

21

depurination

- free radicals damage bases and cause separate of base from sugar
- problems arise during DNA replication

22

deamination of cytosine results in

- uracil
- DNA pol recognizes as thymine

23

nitric oxide or nitrous acid

removes amine group

24

oxidative damage

- causes free radicals that damage DNA
- hydroxyl group backs DNA backbone
- done by hydrogen peroxide (H2O2)
- Guanine to 8-oxo-guanine

25

DNA pol puts what opposite of 8-oxo-G?

- Adenine
- twisted 8-oxo-G looks like thymine

26

Photolyse

- binds to dimer and activated by visible light (340-400 nm)
- uses energy of photon to resolve the pyrimidine dimer (monomerize dimer)
- excision repair
- recombination repair

27

Nucleotide excision repair mediated by UvrABCD

- UvrAB detects error
- UvrC is recruited to the error
- UvrC makes single stranded nick 8 bases 5' to error and 4 bases 3' to error
- UvrD (helicase) releases the 12 base region
- Gap filled and ligated with DNA pol and ligase

28

methyl directed mismatch repair

- involves MutSLH system
- takes advantage of dam methylase to repair misincorporated based in new DNA - slow to methylate all GATC sites in genome

29

steps in methyl directed mismatch repair

- MutS binds to single base pair mismatches
- MutL and MutH are then recruited to the mismatch
- DNA is then looped through until a hemimethylated dam site (GATC) is encountered
- MutH nicks the unmethylated strand - cuts backbone
- MutL separates strand back to mismatch
- DNA is unwound by UvrD and strand is chewed up
- The gap is repaired by DNA pol I and ligase

30

DNA glycosylases

- cleave the sugar bond in altered or damaged nucleotides
- recognize and remove altered or damaged nucleotides
- nick filled by DNA pol 1 and sealed by DNA ligase

31

Daughter strand repair

- replication blocked by pyrimidine dimer which leaves a gap
- the daughter strand that is correct donates its correct strand to the other daughter strand with gap at the T-T dimer.
- mediated by recABC

32

SOS response

- DNA damage detected by RecA protein
- Halts cell division - gives cells time to repair chromosome
- induction of Polymerase V

33

Polymerase V

- can polymerize damaged DNA "translesion"
- not as picky about inserting correct base as pol III
- makes the most mistakes

34

good way to make mutants

- random point mutants - need a screenable phenotype
- treat with mutagen, select for phenotypes - difficult to determine where mutation has taken place

35

better ways to make mutants

- signature tagged mutants
- transposable element mutation - insert randomly in gene and cause mutation. sequence known so DNA elements cloned quickly and insertion site easily identified.

36

best way to make mutants

- gene directed mutation

37

UV light

cross links base pairs

38

ionizing radiation

breaks phosphate backbone

39

nitrous acid

deamination, altered base pairing

40

H2O2

oxidative damage of bases

41

intercolating agents

alter DNA causing base misincorporation
- ethidium bromide

42

screenable phenotypes

- temp sensitivity, motility, antibiotic resistance

43

physiological phenotype

- wild type - prototroph - can make histidine
- mutant - auxotroph - required histidine to grow

44

replica plating

- spread mutagenized cultures on plate where it'll grow
- transfer colonies to 2 plate - 1 with nutrient; 1 without nutrient
- look for colony on plate with nutrient but absent in other.

45

example of auxotrophic mutant

For example, there are auxotrophs for histidine. wild-type strain can make histidine and grow on glucose as sole carbon source.
- if the mutation is in histidinol dehydrogenase, the bacterium requires histidine to be added to the medium to grow.

46

transposable elements

- discrete DNA sequences that encode the proteins necessary for their movement from one site to another
- cause large insertion in coding region that results in a mutation
- known DNA sequence so easy to know where they went

47

gene encoding transposase

- cut out element from one site and integrate into a new site

48

required for transposition

- gene encoding transposase
- terminal target sites recognized by transposase

49

three classes of transposable elements in E. coli

- insertion sequences
- transposons
- bacteriophage Mu

50

insertion sequences

- a transposase gene flanked by inverted repeats
- simplest form

51

transposons

encode a transposase as well as other genes such as drug resistance

52

bacteriophage Mu

- a lysogenic bacteriophage that exclusively uses transposition to replicate.

53

Transposase mechanism

- transposase (as dimer) recognizes inverted repeats at ends and binds, bringing them together forming paired end complex
- double strand DNA cleavage at target sites
- element moves as paired end circular structure to new sequence
- cuts at target sequence and inserts at new sequence.

54

Why use transpose mutagenesis

- insertions can be isolated at a large number of sites on the bacterial chromosome - almost any site mutated
- insertion mutants can be recovered at high frequency
- a transposon insertion in a gene usually causes complete loss of function
- the phenotype of the insertion mutation is completely linked to antibiotic resistance in genetic crosses
- special transposons can be used to construct operon or gene fusions to reporter genes.