Exam 3 Flashcards
mutation
definition
an heritable change in genetic material
mutations positives and negatives
- positive: give allelic variation, foundation for evoluntionary change
- negatives: new mutations more likely to be harmful than beneficial, cause diseases
point mutation types
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)
transitions are _ common that transversions
more
transitions is a change
of a pyrimidine to another or a purine to another
transversion is a change of a
pyrimidine to a purine or vice versa
silent mutations
def
base subsitutions that do not alter the amino acid sequence of the polypeptide
silent mutations are possible because of
the degeneracy of the genetic code
missense mutations
def
base subsitutions where an amino acid sequence change occurs
nonsense mutations
base subsitutions that change a normal codon to a stop codon
frameshift mutations
def
addition or deletion that is not a factor of 3
silent mutations have _ effect on protein function
no
missense mutations have a _ effect on protein function
neutral or inhibitory
up promoter mutations…
increase transcription
a mutation in the promoter..
affects transcription
mutation in the regulatory element or operator site
disrupt the ability of the gene to be properly regulated
mutation in UTR
alter the ability of mRNA to be translated, or alter mRNA stability
mutation in the splice recognition sequence
effect
alter the ability of pre-mRNA to be properly spliced
forward mutation changes
the wild-type genotype into something
reverse mutation changes
a mutant allele back to the wild type
mutations are often characterized by
their differential ability to survive
suppressor mutations
def
reverse the phenotypic effect of another mutation
intragenic suppressors
def
- 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
intergenic supressors
def
- 2nd mutation is different gene than the first mutation
position effect
when a chromosome rearranges and a gene gets moved so the expression is altered
two common reasons for position effects
- movement to a position next to regulatory sequences
- movement to a hetrochromatic regions
animal cell two types
- germ line cells
- somatic cells
germ-line mutations are those that
occur directly in a sperm or egg cell, or in one of the precursor cells
somatic mutations are that occur
directly in a body cell that is not part of the germ-line
_ mutations are found in the whole body
germ-line mutations
somatic mutations result in _
patches of affected area
* earlier the mutation the larger the patch
_ mutations are passed onto gametes
germ line mutations
half the gametes
lamarck proposed that
physiological events determine whether traits are passed along to offspring
lederberg study objective was to
see if mutations are randomly occuring
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
lederberg experiment steps
- place bacteria on plate
- allow cells to divide
- transfer replicas from master plate to secondary plates containing t1 phages
results of lederberg experiment
mutations had occured randomly in absence of selection by T1
* no new colonies in the presence of T1
spotaneous mutations
- result from abnomalities in the biological processes
induced mutations
def
caused by enviromental agents
mutagens
def
agents that alter DNA structure
spontaneous mutations arise from:
3 types
- depurination
- deamination
- tautomeric shift
depurination is the
removal of a purine (A G) from DNA
apurinic site is formed by
depurination
how does depurination cause mutation
sometimes repair system fails and the DNA polymerase adds a random base in the apurinic site (base missing there)
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
deamination happens most with
cytosine
deamination of cytosine turns it into
uracil
_ is a hot spot for mutation
mythylated cytosine bases
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
to cause mutation, a tautomeric shift
must occur immediately prior to replication
oxidative stress is
an imbalance between the production of ROS and organism’s ability to break them down
ROS accumulation can cause
DNA damage and mutation
CAG repeat in coding sequence can cause
long tracks of glutamine that causes proteins to aggregate with each other
TNRE in non conding regions can
- cause abnormal changes in RNA structure
- methylated CpG islands (silence genes)
anticipation
TNRE occur more frequencly during gamete formation, so gets worse with each generation
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
chemical mutagen three types
- base modifiers
- intercalating agents (interfere with replication)
- base analogues
base modifiers do what
covalently modify structure NT
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
x ray and gamma rats have
shirt wavelngths and high energy
ionizing radiation includes
x ray and gamma rays
nonionizing radiation includes
UV light
UV light causes the formation of
cross linked thymine dimers which can cause mutations when replicated
mutation rate
likelihood that a gene will be altered by a new mutation
mutation frequency
the number of mutant genes divided by the total number of genes in a population
mutation frequency can get higher than mutation rates because of
natural selection and genetic drift
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
_ can repair thymine dimers
photolyase
alkyltransferase repairs
alkylated bases by transfering the methyl or ethyl group from the base to a cysteine side chain within the alkyltransferase
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
base excision repair steps
- N glycosylase recognize abnormal base and cleaves bond b/w base and sugar
- AP endonuclease cleaves DNA on 5’ end of missing base
- Creates nick in DNA
- ECOLI: DNA pol 1 removes damaged region and fills normal DNA, ligase seals
- EUKAR: DNA pol beta can fill base OR DNA pol delta/episilon do flap technique, ligase seals
base excision repair is for
small repairs, one nucleotide
nucleotide excision repair can repair
many types of DNA damage, more major problems
nucleotide excision repair steps
- UvrA/B tracks along DNA in search
- After damage detected, UvrA released and UvrC binds
- UvrC makes cuts on both sides of the damage
- UvrD removes the damaged regions
- UvrB&C released
- DNA pol fills in the gaps, ligase seals
mutations in genes involving nucleotide excision repair all cause diseases that
increase sensitivity to sunlight
DNA pol have _ to recgonize _ and repair them
3’ to 5’ proofreading activity to detect base mismatches and fix them
mis match repair system is specific
to the newly made daughter strand
how does mismatch repair system know difference between parent and daughter strand
parent strand is methylated while daughter is not
mismatch repair system steps
- MutS finds mismatch
- MutS and L bind to MutH which is already bound to a sequence
- MutH cuts nonmethylated strand
- MutU seperates DNA
- Endonuclease digest DNA to point where MutS is binded
- Base mitchmatach is yeeted
- DNA pol fill and ligase seals
homologous recombination is only available during _ because _
during S and G2 pahses because sister chromatid is needed
homologous recombination is error_ because
error free because sister chromatids are genetically identical
homologous recombination steps
- end processing of broken strand (ends are chewed in)
- strand exchange
- non-broken strands act as a template for fixing thhe break
- resolution by cutting strands and ligase reattching, part of unbroken strand now on fixed strand
non homologous end joining is error_ because
error prone because processing may result in a deletion
nonhomologous end joining steps
- end binding proteins bind to each broken end
- other proteins form a cross bridge
- gap is repaired and ligase seal
DNA pol 3 in e coli is unable to
replicate lesioned DNA
translesion synthesis is possibel because
translesion-replicating pol contain an active site with a loose pocket so can accomodate weird structures
a negative to the translesion synthesis process
translesion -replicating pol have low fidelity, high mutation rate
homologous recombination occurs in
meiosis 1
homologous recombination involves
alignment of pair of chromosomes, breakage at analogous locations and exchange of segments
homologous recombination steps
holiday model
- both chromosomes nicked at identical locations
- DNA strands to the left of the nicks invade and link to the strands to the right of the nicks
- creates a holiday junction
- holiday junction migrates (brand migration)
- creates two hetroduplex regions
- hetroduplex regions have some base mismatches
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
gene conversion
two different alleles become one
gene conversion occurs
- dna mismatch repair
- DNA gap repair synthesis: double stranded break area loses a small part
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
What is a key characteristic(s) of the repeat sequences that promote trinucleotide repeat expansion?
contain a C and G
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+
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
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
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
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
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
5-bromouracil would cause what type of mutation
transition mutation
thymine analoug
problem set 10
what type of mutation would nitrous acid make
transition mutation
PS 10
what type of mutation proflavin cause
frameshift mutation
intercalating agent
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.
recombinant DNA technology is
the use of in vitro molecular techniques to isolate and manipulate fragments of DNA
process of cloning genes into vectors
- endonucleases cut genes into sticky ends that can base pair to another DNA sequence with complementary sequence
- other DNA is cut with the same enzyme
- incubate the DNA together
- sticky ends hydrogen bond
- add DNA ligase
- result can be a recirculized vector, recombinant with target or recombinant without target gene
- test to see which vector has target gene
- if recombinant with right target, gene will be white and beta galac inactive
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
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
sticky ends are most stable when they
have more C and Gs
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
amplification of a clones genes occurs in two ways
- vector gets replicated by host many times
- bacterial cell divides approx every 20 mins
complementary DNA
DNA made from RNA
cDNA has
only the coding parts of the gene
* allows expression of the encoded protein
how is cDNA made
- add poly-dT primer to mRNA that binds poly A tail of mRNA
- add reverse transcriptase and dNTPs
- add RNaseH to cut up the mRNA template and make RNA primers
- add DNA pol 1 and DNA ligase to synthesize the second DNA strand
genomic library
contains whole genomes, starting material is chromosomal DNA
* contains colonies of dna that carry seperate peices of chromosomal DNA
cDNA library
starting material is cDNA
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
making cDNA library
- isolate mRNA
- make cDNA
- add linker DNA that is a sequence recognized by restriction enzymes
- cut cDNA and plasmid DNA with RE and ligate cDNA into vectors
- transform bacteria
- each colony has cDNA peice
PCR requires enough
knowledge of the gene to have short primers
starting material for PCR
- template DNA
- oligonucleotide primers
- dNTPs
- Taq polymerase
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.
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.
PCR cycle steps
- Denaturation- DNA strands are seperated with high temp
- Primer annealing- Primers bind to DNA strands *lower temp *
- Primer extension nucleotides are added to the primers, synthesis of complementary strands slightly higher temp
after many cycles of PCR
there will be fragments that only contain the region of interest and many copies of it
_ can be used to amplify small samples
PCR
reverse transcriptase PCR
used to amplify RNA
reverse transcriptase PCR steps
- add reverse transcriptase to RNA along with primer at 3’ end and dNTPs
- make cDNA
- do normal PCR with the cDNA
reverse transcriptase PCR is _ specific
highly
RT PCR is used to
quantitate the amount of specfic gene or mRNA in a sample
how does RT PCR work
- Taqman Probe is complementary to PCR product, has reporter fluorenscent molecule on one side and quencher that absorbs the fluorenscents
- during primer annealing, both primer and TaqMan bind to DNA
- Taw polymerase digests the TawMan probe which seperates the reporter and quencher
- reporter fluorescence is now detected
- see fluorescence = strand has been made
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.
phases of fluorescence in RT PCR
first linear, then pass the cycle threshold and linear growth then plateau
chain termination
ddNTPs added to growing DNA strand so the strand can no longer grow
dideoxy sequencing
- make single stranded recombinant vector
- mix recombinant vector and primer
- add normal dNTPs alot
- add fluoresc ddNTPs a little
- add DNA pol
- strands made with ddNTPs chain terminating
- seperate sequences by length
- use colors to derieve sequence
site directed mutagenesis allows researchers to see how
mutations affect:
* expression
* function of a protein
* phenotype of an organism
Cas-CRSPR explained
- sgRNA has the crRNA with the spacer and repeat, linker, tracrRNA
- spacer of sgRNA binds to complementary region of target gene
- cas9 cleaves the target gene in both strands
- nonhomologous end joinin has no donor so creates small deletion in gene
- homologous recombination repair uses donor and creates point mutation/point fixing
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
northern blotting is used to identify
a specific RNA within a mixture of many RNA molecules
northern blotting procedure
- a mizture containing many different RNAs is extracted from cells
- the RNAs are seperated from each other by gel electrophoresis
- the RNAs in the gel are then blotted onto a nitrocellulose or nylon filter
- the filter is placed into a soliution containing a labeled probe
- the RNA that is complementary to the labeled probe is the one detected as a dark band
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
western blotting is used to
detect a specific protein
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
