Bacterial Genetics 1 Flashcards
1
Q
The best way to see what a gene does
A
- break it and see what happens
2
Q
the best way to tell what gene controls a process
A
break lots of genes and look for an organism that can’t do the process
3
Q
mutation
A
change in DNA sequence
4
Q
isogenic
A
all genes except one are the same
5
Q
species
A
a population of microorganisms with similar characteristics
6
Q
clone
A
a population of cells that are genetically identical
7
Q
genotype
A
the specific set of genes present in a cell
8
Q
phenotype
A
the collection of characteristics that are available
9
Q
wild type strain
A
- a recognized “type” strain, which is a clonal population of a particular species
- has the identical genotype and phenotype
10
Q
mutagens
A
- chemicals
- ionizing radiation
- UV radiation
- agents that increase mistakes in DNA replication
11
Q
point mutation
A
- change of one base to another
12
Q
point mutation outside of protein coding region
A
- no effect
13
Q
silent mutation
A
codon still encodes same AA
14
Q
missense mutation
A
codon codes for different AA
15
Q
read through mutation
A
- normal termination sequence is mutated and no longer stops translation
16
Q
nonsense mutation
A
change to termination codon.
17
Q
frameshift mutation
A
- if one of two bases added or deleted within the coding region
- new AA sequence downstream
18
Q
spontaneous mutations
A
- 1 mutation/generation
- 10^-6 to 10^-8 per generation
- errors not fixed in DNA replication
- DNA damage
19
Q
UV induced damage
A
- 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
Q
ionizing radiation
A
- radiation forms chemical free radical, which can cause double strand breaks in DNA
21
Q
depurination
A
- free radicals damage bases and cause separate of base from sugar
- problems arise during DNA replication
22
Q
deamination of cytosine results in
A
- uracil
- DNA pol recognizes as thymine
23
Q
nitric oxide or nitrous acid
A
removes amine group
24
Q
oxidative damage
A
- 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.
