Exam 3 Flashcards
(143 cards)
1
Q
mutation
definition
A
an heritable change in genetic material
2
Q
mutations positives and negatives
A
- positive: give allelic variation, foundation for evoluntionary change
- negatives: new mutations more likely to be harmful than beneficial, cause diseases
3
Q
point mutation types
A
base subsitions two types:
* transition (change within pyrimidine and purine (C ⇔ T OR A ⇔ G)
* transversion (change b/w pyrimidine and purine (C,T ⇔ A,G)
4
Q
transitions are _ common that transversions
A
more
5
Q
transitions is a change
A
of a pyrimidine to another or a purine to another
6
Q
transversion is a change of a
A
pyrimidine to a purine or vice versa
7
Q
silent mutations
def
A
base subsitutions that do not alter the amino acid sequence of the polypeptide
8
Q
silent mutations are possible because of
A
the degeneracy of the genetic code
9
Q
missense mutations
def
A
base subsitutions where an amino acid sequence change occurs
10
Q
nonsense mutations
A
base subsitutions that change a normal codon to a stop codon
11
Q
frameshift mutations
def
A
addition or deletion that is not a factor of 3
12
Q
silent mutations have _ effect on protein function
A
no
13
Q
missense mutations have a _ effect on protein function
A
neutral or inhibitory
14
Q
up promoter mutations…
A
increase transcription
15
Q
a mutation in the promoter..
A
affects transcription
16
Q
mutation in the regulatory element or operator site
A
disrupt the ability of the gene to be properly regulated
17
Q
mutation in UTR
A
alter the ability of mRNA to be translated, or alter mRNA stability
18
Q
mutation in the splice recognition sequence
effect
A
alter the ability of pre-mRNA to be properly spliced
19
Q
forward mutation changes
A
the wild-type genotype into something
20
Q
reverse mutation changes
A
a mutant allele back to the wild type
21
Q
mutations are often characterized by
A
their differential ability to survive
22
Q
suppressor mutations
def
A
reverse the phenotypic effect of another mutation
23
Q
intragenic suppressors
def
A
- the second mutation is within the same gene as the first mutation
- typically the first mutations causes abnomality in protein and the second mutation restores normal protein structure
24
Q
intergenic supressors
def
A
- 2nd mutation is different gene than the first mutation
25
position effect
when a chromosome rearranges and a gene gets moved so the expression is altered
26
two common reasons for position effects
1. movement to a position next to regulatory sequences
2. movement to a hetrochromatic regions
27
animal cell two types
* germ line cells
* somatic cells
28
germ-line mutations are those that
occur directly in a sperm or egg cell, or in one of the precursor cells
29
somatic mutations are that occur
directly in a body cell that is not part of the germ-line
30
_ mutations are found in the whole body
germ-line mutations
31
somatic mutations result in _
patches of affected area
* earlier the mutation the larger the patch
32
_ mutations are passed onto gametes
germ line mutations
| half the gametes
33
lamarck proposed that
physiological events determine whether traits are passed along to offspring
34
lederberg study objective was to
see if mutations are randomly occuring
35
in the lederberg experiment, if the physiological adaptation hypothesis is true,
then the number of tonr bacteria would be low unless there is selection for T1 resistance
36
lederberg experiment steps
1. place bacteria on plate
2. allow cells to divide
3. transfer replicas from master plate to secondary plates containing t1 phages
37
results of lederberg experiment
mutations had occured randomly in absence of selection by T1
* no new colonies in the presence of T1
38
spotaneous mutations
* result from abnomalities in the biological processes
39
induced mutations
| def
caused by enviromental agents
40
mutagens
| def
agents that alter DNA structure
41
spontaneous mutations arise from:
| 3 types
* depurination
* deamination
* tautomeric shift
42
depurination is the
removal of a purine (A G) from DNA
43
apurinic site is formed by
depurination
44
how does depurination cause mutation
sometimes repair system fails and the DNA polymerase adds a random base in the apurinic site (base missing there)
45
depurination has a _ chance of mutation in _one or both_ daughter strands
75% of mutation in one of the daughter strand
| wrong base can be filled in the a puranic site
46
deamination happens most with
cytosine
47
deamination of cytosine turns it into
uracil
48
_ is a hot spot for mutation
mythylated cytosine bases
49
tautomeric shift is when
thymine and guanine go from keto form to being in enol form
A and C convert from amino to imino form
50
to cause mutation, a tautomeric shift
must occur immediately prior to replication
51
oxidative stress is
an imbalance between the production of ROS and organism's ability to break them down
52
ROS accumulation can cause
DNA damage and mutation
53
CAG repeat in coding sequence can cause
long tracks of glutamine that causes proteins to aggregate with each other
54
TNRE in non conding regions can
* cause abnormal changes in RNA structure
* methylated CpG islands (silence genes)
55
anticipation
TNRE occur more frequencly during gamete formation, so gets worse with each generation
56
mechanism of TNRE
* TNRE contain C and G that create hairpin
* polymerase slip off DNA, hairpin forms
* DNA pol hops back on and beings synthesis from new location
* hairpin spreads out, the gaps are filled by gap repair
57
chemical mutagen three types
* base modifiers
* intercalating agents (interfere with replication)
* base analogues
58
base modifiers do what
covalently modify structure NT
59
intercalating agents
contain flat planar structures that intercalate themselves in the double helix
* causes distortion of helix
* daughter strand may have additions or deletions
* frame shifts
60
x ray and gamma rats have
shirt wavelngths and high energy
61
ionizing radiation includes
x ray and gamma rays
62
nonionizing radiation includes
UV light
63
UV light causes the formation of
cross linked thymine dimers which can cause mutations when replicated
64
mutation rate
likelihood that a gene will be altered by a new mutation
65
mutation frequency
the number of mutant genes divided by the total number of genes in a population
66
mutation frequency can get higher than mutation rates because of
natural selection and genetic drift
67
how does the ames test work
* a strain of bacteria that cannot make histindine is used bc of a point mutation
* a second mutation may occur to reverse this
* ames test monitors the rate at which the second mutation occurs
68
_ can repair thymine dimers
photolyase
69
alkyltransferase repairs
alkylated bases by transfering the methyl or ethyl group from the base to a cysteine side chain within the alkyltransferase
70
base excision repair involves _ that can _
DNA N-glycosylases that can recognize an abnormal base and cleave the bond between it and the sugar in the DNA
71
base excision repair steps
1. N glycosylase recognize abnormal base and cleaves bond b/w base and sugar
2. AP endonuclease cleaves DNA on 5' end of missing base
3. Creates nick in DNA
4. ECOLI: DNA pol 1 removes damaged region and fills normal DNA, ligase seals
4. EUKAR: DNA pol beta can fill base OR DNA pol delta/episilon do flap technique, ligase seals
72
base excision repair is for
small repairs, one nucleotide
73
nucleotide excision repair can repair
many types of DNA damage, more major problems
74
nucleotide excision repair steps
1. UvrA/B tracks along DNA in search
2. After damage detected, UvrA released and UvrC binds
3. UvrC makes cuts on both sides of the damage
4. UvrD removes the damaged regions
5. UvrB&C released
6. DNA pol fills in the gaps, ligase seals
75
mutations in genes involving nucleotide excision repair all cause diseases that
increase sensitivity to sunlight
76
DNA pol have _ to recgonize _ and repair them
3' to 5' proofreading activity to detect base mismatches and fix them
77
mis match repair system is specific
to the newly made daughter strand
78
how does mismatch repair system know difference between parent and daughter strand
parent strand is methylated while daughter is not
79
mismatch repair system steps
1. MutS finds mismatch
2. MutS and L bind to MutH which is already bound to a sequence
3. MutH cuts nonmethylated strand
4. MutU seperates DNA
5. Endonuclease digest DNA to point where MutS is binded
6. Base mitchmatach is yeeted
7. DNA pol fill and ligase seals
80
homologous recombination is only available during _ because _
during S and G2 pahses because sister chromatid is needed
81
homologous recombination is error_ because
error free because sister chromatids are genetically identical
82
homologous recombination steps
1. end processing of broken strand (ends are chewed in)
2. strand exchange
3. non-broken strands act as a template for fixing thhe break
4. resolution by cutting strands and ligase reattching, part of unbroken strand now on fixed strand
83
non homologous end joining is error_ because
error prone because processing may result in a deletion
84
nonhomologous end joining steps
1. end binding proteins bind to each broken end
2. other proteins form a **cross bridge**
3. gap is repaired and ligase seal
85
DNA pol 3 in e coli is unable to
replicate lesioned DNA
86
translesion synthesis is possibel because
translesion-replicating pol contain an active site with a loose pocket so can accomodate weird structures
87
a negative to the translesion synthesis process
translesion -replicating pol have low fidelity, high mutation rate
88
homologous recombination occurs in
meiosis 1
89
homologous recombination involves
alignment of pair of chromosomes, breakage at analogous locations and exchange of segments
90
homologous recombination steps
| holiday model
1. both chromosomes nicked at identical locations
2. DNA strands to the left of the nicks invade and link to the strands to the right of the nicks
3. creates a holiday junction
4. holiday junction migrates (*brand migration*)
5. creates two hetroduplex regions
6. hetroduplex regions have some base mismatches
91
recent models of recombination steps
* no nick in the same location
1. DNA helix incur break in both strands of one chromatid
2. Ends processed
2. strand invasion fors D-loop
3. gap repair synthesis fills in
4. branch migration
92
gene conversion
two different alleles become one
93
gene conversion occurs
1. dna mismatch repair
2. DNA gap repair synthesis: *double stranded break area loses a small part*
94
A mutation changes a codon to another codon that does not change the original amino acid to a different amino acid. This is a
silent mutation
95
What is a key characteristic(s) of the repeat sequences that promote trinucleotide repeat expansion?
contain a C and G
96
During the Ames test, if a substance is a mutagen, there will be_________ colonies on the plates in which the cells had been exposed to the mutagen, because the mutagen converts some of the cells from ____________.
more, his- to his+
97
During nucleotide excision repair, which of the following does not happen?
* detection of a DNA lesion
* making cuts on either side of the lesion in the same strand
* removing the damaged strand
* making a double stranded cut in the DNA
* making a double stranded cut in the DNA
98
During nucleotide excision repair, which of the following does not happen?
* detection of a DNA lesion
* making cuts on either side of the lesion in the same strand
* removing the damaged strand
* making a double stranded cut in the DNA
* making a double stranded cut in the DNA
99
A gene exists in two alleles, which we will call B and b. The gene is 1123 bp in length, and the B and b alleles exhibit single base pair differences at six different sites. If gene conversion changed the b allele into the B allele, which mechanism would you favor to explain the conversion?
gap repair synthesis
100
According to the double-strand break model for homologous recombination, what happens right after DNA strand degradation at the double-stranded break site?
formation of a D loop
101
According to the Holliday model for homologous recombination, what is/are the possible end result(s) of the resolution step?
nonrecombinant or recombinant chromosomes with a heteroduplex region
102
5-bromouracil would cause what type of mutation
transition mutation
| thymine analoug
## Footnote
problem set 10
103
what type of mutation would nitrous acid make
transition mutation
## Footnote
PS 10
104
what type of mutation proflavin cause
frameshift mutation
| intercalating agent
105
A mutagen changes cytosine to uracil. Chemically, what is the mutagen doing to cytosine to convert it to uracil? Give an example of a mutagen that can do this. Which DNA repair system(s) would be able to repair this defect?
This spontaneous mutation is called a deamination of cytosine which changes a cytosine to a uracil. An amino group is removed from the cytosine which makes it a uracil and the DNA repair enzyme removes it because uracil is not found in the DNA sequence. NH2 in the base is switched into an O.
106
recombinant DNA technology is
the use of in vitro molecular techniques to isolate and manipulate fragments of DNA
107
process of cloning genes into vectors
1. endonucleases cut genes into sticky ends that can base pair to another DNA sequence with complementary sequence
2. other DNA is cut with the same enzyme
3. incubate the DNA together
4. sticky ends hydrogen bond
5. add DNA ligase
6. result can be a recirculized vector, recombinant with target or recombinant without target gene
7. test to see which vector has target gene
8. if recombinant with right target, gene will be white and beta galac inactive
108
when insert gene, it has ampr and lacz why
* amp r shows that cells have taken up the vector
* lax z shows the vector is the targetted gene one
109
As described in your textbook, one way to determine if a segment of chromosomal DNA has been inserted into a vector is to put XGal in the growth media. Following overnight growth...
white colonies contain a vector with an inserted fragment of chromosomal DNA; the lacZ gene has been inactivated
110
sticky ends are most stable when they
have more C and Gs
111
how is functionality of the Lac Z gene determined in recombinant vectors?
X-gal is cleaved by beta galactosidase into a blue dye
* so white colonies have a inactive lax z due to insertation of a DNA fragment
112
amplification of a clones genes occurs in two ways
* vector gets replicated by host many times
* bacterial cell divides approx every 20 mins
113
complementary DNA
DNA made from RNA
114
cDNA has
only the coding parts of the gene
* allows expression of the encoded protein
115
how is cDNA made
1. add poly-dT primer to mRNA that binds poly A tail of mRNA
2. add reverse transcriptase and dNTPs
3. add RNaseH to cut up the mRNA template and make RNA primers
4. add DNA pol 1 and DNA ligase to synthesize the second DNA strand
116
genomic library
contains whole genomes, starting material is chromosomal DNA
* contains colonies of dna that carry seperate peices of chromosomal DNA
117
cDNA library
starting material is cDNA
118
how are genomic libraries made
* create hybrid vectors (technique of recombi vectors)
* transform plasmid vectors into DNA
* each bacterial colony holds a different peice of chromosomal DNA
119
making cDNA library
1. isolate mRNA
2. make cDNA
3. add linker DNA that is a sequence recognized by restriction enzymes
4. cut cDNA and plasmid DNA with RE and ligate cDNA into vectors
5. transform bacteria
6. each colony has cDNA peice
120
PCR requires enough
knowledge of the gene to have short primers
121
starting material for PCR
* template DNA
* oligonucleotide primers
* dNTPs
* Taq polymerase
122
Why is Taq polymerase used in PCR as opposed to DNA polymerase from another source such as E. coli?
Taq polymerase is resistant to heat denaturation.
123
Why is Taq polymerase used in PCR as opposed to DNA polymerase from another source such as E. coli?
Taq polymerase is resistant to heat denaturation.
124
PCR cycle steps
1. **Denaturation**- DNA strands are seperated with *high temp*
2. **Primer annealing**- Primers bind to DNA strands *lower temp *
3. **Primer extension** nucleotides are added to the primers, synthesis of complementary strands *slightly higher temp*
125
after many cycles of PCR
there will be fragments that only contain the region of interest and many copies of it
126
_ can be used to amplify small samples
PCR
127
reverse transcriptase PCR
used to amplify RNA
128
reverse transcriptase PCR steps
1. add reverse transcriptase to RNA along with primer at 3' end and dNTPs
2. make cDNA
3. do normal PCR with the cDNA
129
reverse transcriptase PCR is _ specific
highly
130
RT PCR is used to
quantitate the amount of specfic gene or mRNA in a sample
131
how does RT PCR work
1. Taqman Probe is complementary to PCR product, has reporter fluorenscent molecule on one side and quencher that absorbs the fluorenscents
2. during primer annealing, both primer and TaqMan bind to DNA
3. Taw polymerase digests the TawMan probe which seperates the reporter and quencher
4. reporter fluorescence is now detected
5. see fluorescence = strand has been made
132
During real-time PCR, why does the level of fluorescence given off by the TaqMan probe increase over time?
Taq polymerase cleaves the TaqMan probe, which separates the quencher and the reporter.
133
phases of fluorescence in RT PCR
first linear, then pass the cycle threshold and linear growth then plateau
134
chain termination
ddNTPs added to growing DNA strand so the strand can no longer grow
135
dideoxy sequencing
1. make single stranded recombinant vector
2. mix recombinant vector and primer
3. add normal dNTPs alot
4. add fluoresc ddNTPs a little
5. add DNA pol
6. strands made with ddNTPs chain terminating
7. seperate sequences by length
8. use colors to derieve sequence
136
site directed mutagenesis allows researchers to see how
mutations affect:
* expression
* function of a protein
* phenotype of an organism
137
Cas-CRSPR explained
1. sgRNA has the crRNA with the spacer and repeat, linker, tracrRNA
2. spacer of sgRNA binds to complementary region of target gene
3. cas9 cleaves the target gene in both strands
4. nonhomologous end joinin has no donor so creates small deletion in gene
5. homologous recombination repair uses donor and creates point mutation/point fixing
138
Which component of the CRISP-Cas system is needed if the goal is to create a point mutation (such as changing one codon into a different codon) in a target gene, but it is not needed to create a deletion in a target gene?
donor DNA
139
northern blotting is used to identify
a specific RNA within a mixture of many RNA molecules
140
northern blotting procedure
1. a mizture containing many different RNAs is extracted from cells
2. the RNAs are seperated from each other by gel electrophoresis
3. the RNAs in the gel are then blotted onto a nitrocellulose or nylon filter
4. the filter is placed into a soliution containing a labeled probe
5. the RNA that is complementary to the labeled probe is the one detected as a dark band
141
interpreting northern blots
how far up the line shows how much expressed/made in cell
* thickness of band shows molecular weights
* three different molecular weights means the mRNA is alternatively spliced
142
western blotting is used to
detect a specific protein
143
procedure of western blotting
mizture of many different proteins are extracted from cells and seperated by SDS Page
* protein bands within the gel are blotted onto a nitrocellulose or nylon filter
* filter placed in a primary antibody that recognizes protein of interest
* secondary antibody conjugated to alkaline phosphatase recignizes the constant region of the primary antibody
* colorless dye added
* alkaline phospha make dye black
* protein of interst revealed as a dark band
