genetics exam 4 Flashcards
what is epigenetics?
the transmission of info from one cell generation to the next without altering/involving DNA sequences
changes in gene expression that can be passed from cell to cell and are reversible but DOES NOT involving changing DNA seq
what are epigenetics effects due to?
- histone modifications/nucleosome remodeling (involves methylation and acetylation)
- DNA methylation
what is significant about DNA methylation?
DNA methylation is HERITABLE
methylated DNA seqs are inherited during cell division (daughter cells carry the same modification patterns as the mother cell)
pattern of one copy of gene being methylated and the other not is maintained in resulting offspring
explain the molecular model for inheritance of DNA methylation
de novo methylation is an INFREQUENT and HIGHLY REGULATED event
begin with parent’s methylated DNA –> undergoes DNA replication where each daughter DNA strand gets one of the parent’s methylated DNA, making hemimethylated DNA strands –> maintenance methylase targets hemimethylated DNA and will methylated the unmethylated daughter strands
explain modified histone inheritance of cells from generation to generation
old modified histones distributed randomly among 2 daughter DNA molecules
old modified histones serve as TEMPLATES for mod of new histones
self-perpetuating state
epigenetic inheritance = patterns of chromatin mod are passed onto next cell generation
how are genes/chromosomes targeted for epigenetic regulation?
- targeting gene for epigenetic mod by a TRANSCRIPTION FACTOR –> transcription factor recognizes specific gene sequences and binds to them, transcription factor will recruit other proteins (like histone-modifying enzymes and DNA methyltransferase) which will lead to changes in chromatin structure or DNA methylation, changes alter expression of gene and are maintained in subsequent cell divisions
- targeting gene for epigenetic mod by NONCODING RNA –> non-coding RNA recognizes specific gene sequences and binds to them, non-coding RNA will recruit other proteins (like histone-mod enzymes and DNA methyltransferase) which leads to changes in chromatin structure or DNA methylation, changes alter expression of this gene and are maintained in subsequent cell divisions
what are the 2 categories of epigenetic gene regulation?
- during the life of the organism = epigenetic gene regulation or epigenetic marks may occur as a programmed developmental change (cell differentiation)
- trans-generational = epigenetic patterns inherited from parents
genomic imprinting = one copy of a gene is INHERITED IN AN INACTIVE state
what can result in epigenetic changes?
environmental agents like temp, diet, toxins
these can alter epigenetic marks
what are the mechanisms to maintain epigenetic marks?
- DNA methylation = hemimethylated DNA becomes fully methylated via maintenance methylation
- histone modifications = histones recruit chromatin-mod enzymes and chromatin-remodeling complexes to daughter chromatids
why are epigenetic marks important?
they tell our cells whether to turn a gene on or off
when are epigenetic marks of adults erased?
mostly erased at gametogenesis/fertilization
adults have stable epigenetic marks in all cells –> fusion of gametes involves resetting which erase epigenetic marks so that the fertilized egg can develop into any type of cell (few or no epigenetics from parents) –> fetal development is when new germ cells develop in fetus and new epigenetic marks are established as cells differentiate
there are exceptions to this rule like imprinted genes
what is genomic imprinting?
a phenomenon in which a segment of DNA is inherited in a SILENCED STATE (in adult, one copy is silent and the other is active)
depending on how the genes are marked, the offspring expresses either MATERNALLY-inherited or PATERNALLY-inherited allele = monoallelic expression –> even if a person is mut/wt heterzygous, if the wt copy is silenced and only the mut is expressed then the person displays the disease
what are the stages of imprinting?
- establishment of imprint during embryogenesis (in parent)
- maintenance of imprint during embryogenesis and in adult somatic cells (in offspring)
- erasure and re-establishment of imprint in the germ cells of offspring
review slide 16 about Igf2 imprinting lecture 31
explain the stages of imprinting in relation to Igf2
- establishment of imprint
imprinting of Igf2 gene occurs during gametogenesis, sperm carries the Igf2 allele and egg carries Igf2- allele, only paternal allele will be expressed in offspring - maintenance of imprint
after fertilization, imprint pattern is maintained throughout development –> in this example, maternal Igf2- will NOT be expressed in somatic cells - erasure and re-establishment
in germ-line cells, imprint is erased –> female mouse produces eggs in which the gene is silenced (has Igf2 and Igf2- but they’re all silenced) and male produces sperm in which gene can be transcribed into mRNA (has Igf2 and Igf2- that are all transcribed)
what are some examples of human diseases associated with imprinted genes?
Prader-Willi syndrome and Angelman syndrome
how do you differentiate between paternal and maternal imprinting?
paternal imprinting (paternal gene silenced) = paternally-inherited allele is inherited in SILENT state –> half the progeny of affected females will be affected regardless of their gender
ex: if offspring inherits wt or mut from mother then they will pass it on to 50% of their children, but if they inherit wt or mut from father, none of the genes will be expressed (50% offspring will be carriers)
maternal imprinting (maternal gene silenced) = maternally-inherited allele is inherited in a SILENT state –> half the progeny of affected males will be affected regardless of their gender
affected individuals are HETERZYGOTES
SAME GENDER as parents will be carriers while opp genders will pass on to 50% of their offpsring
explain the effects of diet on genetics in mice
researchers fed pregnant YELLOW mouse a METHYL-RICH diet –> most of her pups were brown and stayed healthy for life (lots of methylation)
decrease methylation of agouti gene and only fed with BPA –> her pups were yellow
2 mice have same mutation in agouti gene –> its promoter is VERY SENSITIVE to methylation (when highly expressed (no Me++), agouti affects coat color, obesity, diabetes, and tumorigenesis
what are non-coding RNAs?
some genes DO NOT encode polypeptides, but are transcribed into non-coding RNAs (ncRNAs)
in most cell types, ncRNAs are more abundant than mRNAs (in human cell –> only about 20% of transcription involves production of mRNAs whereas 80% of it is associated with making ncRNAs)
ncRNAs can bind to different types of molecules
RNA molecules can form stem-loop structures which may bind to pockets on surface of proteins
ncRNAs can have multiple binding sites (RNA acts as a scaffold)
what are some common binding interactions between ncRNA and other molecules?
- ncRNA-DNA binding
- ncRNA-protein binding
- ncRNA-mRNA binding
- ncRNA-small molecule binding
what are the functions of ncRNAs?
they can function as:
- scaffold = ncRNA binds a group of proteins
- alteration of protein function or stability (ncRNA binds to a protein and alters that protein’s structure –> ability of protein to act as a catalyst, to bind to another molecule, or stability of the protein)
- guide = ncRNA binds to a protein and guides it to a specific site in the cell
some are housekeeping RNAs like telomerase RNAs, rRNAs, tRNAs, components of splicing machineries, guide RNAs, ribozymes and components of secretory machinery
how does ncRNA act as a guide?
it can direct a protein to a specific DNA/other RNA seq
ncRNA guides protein to a site in the DNA
this is the kind of function that tracrRNA+crRNA provides in the CRISPR/Cas9 system (Cas9 is directed to a particular DNA seq in a DNA molecule by base pairing of guide RNA with the DNA target
what are the ncRNA lengths?
long ncRNAs (lncRNAs) are longer than 200 nt –> this includes many of the housekeeping RNAs and some long, regulatory RNAs
small regulatory RNAs (short ncRNAs) are shorter than 200 nt –> microRNAs, siRNAs and piRNAs fall into this catergory (usually 20-25 nt) –> these 3 classes of sncRNAs silence gene expression
how do small regulatory RNAs (miRNA, piRNA and siRNA) regulate gene expression?
there are 3 mechanisms
- translation repression of target seqs = bind to mRNA and prevent its translation
- mRNA destruction of target seqs = bind to mRNA and target it for destruction
- silencing chromatin = bind to/near the DNA seq and cause it to be heterochromatinized
what is the cellular roles of ncRNAs?
researchers used antisense RNA to inhibit mRNA translation (RNA complementary to mRNA)
researchers also introduced sense RNA which also inhibited mRNA expression (RNA os SAME SEQ and POLARITY as mRNA)
explain the use of mRNA in C. elegans
one experiment involved an mRNA encoded by gene called mex-3
an abundant mRNA in early embryos of C. elegans –> make sense and antisense mex-3 RNA in vitro using cloned genes for mex-3 –> inject either mex-3 antisense RNA or a mix of mex-3 sense and antisense RNA into gonads of C. elegans –> RNA is taken up by eggs and early embryos then you see what happened to the mRNA –> used in situ hybridization for this
how does the experiment mRNA and mex-3 work?
- incubate and then subject early embryos in situ hybridization (a laboratory technique that finds and localizes specific DNA or RNA sequences in biological samples)
- in this method, labeled probe is added that is complementary to mex-3 mRNA
- probe is labeled with green fluorescent dye
- if cells express mex-3, the mRNA in cells will bind to the probe and become labeled with green
- after incubation with a labeled probe, the cells are washed to remove unbound probe
- observe embryos under a microscope
the intensity of green color is proportional to [mRNA] –> control is really green (high levels of mex-3 mRNA), injected with mex-3 antisense RNA is moderately green (mRNA levels decreased), injected with dsRNA (no mRNAs detected)
this data indicates that dsRNA is more POTENT at SILENCING mRNA than antisense RNA = RNA interference (RNAi) which dsRNA causes the silencing of mRNA
what are examples of RNAi?
gene silencing mechanisms mediated by miRNAs, siRNAs, or piRNAs
RNAi is found in most eukaryotic species
- microRNAs (miRNAs) are transcribed from ENDOGENOUS eukaryotic genes (as many as 60% of human genes may be regulated by microRNA)
- small interfering RNAs (siRNAs) include those that originate from EXOGENOUS sources (not normally made by cells and those encoded on genome
- PIWI-interacting RNAs (piRNAs) are less widely distributed, mostly in ANIMAL GONADS and target transposable elements
what is significant about hte 3 types of RNAi?
they differ mostly in ORIGIN/BIOGENESIS
all 3 silence genes, associated with conserved set of proteins and use base complementarity to id mRNA targets
what are small RNAs processed from?
larger precursors
- miRNAs = pri-miRNA is made –> folds up on itself to form a HAIRPIN recognized by RNA processing enzymes, cleaved to 70-nt pre-miRNA and exported, cut by dicer to 20-25 bp
- siRNA (long dsRNA produced) –> these are either encoded or sometimes produced in response to viral infections, processed into smaller fragments by dicer enzymes
- long dsRNAs are artificially introduced into cell will also be processed to siRNAs
what do small RNAs form?
they associate with a group of proteins to form silencing complexes –> RNA-Induced Silencing Complex (RISC) [binds to mRNA and causes degradation] or RITS (RNA-induced initiation of transcriptional silencing) [silences mRNA]
what do RISC and RITS contain?
- dsRNA molecule that is 20-25 bp long produced from pre-miRNAs and pre-siRNAs by dicer (one strand will be degraded and the other will be retained)
- proteins
- piRNAs associate with PIWI proteins to form complexes known as piRNA-induced silencing complexes (piRISCS)
- all complexes are directed to their target site by base pairing with small RNA
review slide 17 of lecture 32
what are the silencing mechanisms of the small RNAs?
siRNA derived from dsRNA of viral replication and miRNA from pre-miRNA (this is easily manipulated) will form RISC or RITS –> RITS or RISC complex is GUIDED to mRNA target by complementarity between small RNA and mRNA
piRNAs cause transcriptional silencing of transposable elements via piRISCs complexes
RITS can lead to DNA/histone methylation while RISC leads to mRNA degradation and translation block
what is the function and benefits of RNAi?
RNAi is important form of GENE REGULATION (silences the expression of specific mRNAs but will NEVER INCREASE gene expression)
widely used by plants to prevent viral infections
siRNAs can be introduced into a cell or produced in cell by genetic engineering to target specific genes for silencing
this is huge therapeutic value
what was the first FDA approved RNA-based therapeutic?
Patisiran is a ds siRNA that specifically binds to a genetically conserved seq in 3’-untranslated region of mut and wt transthyretin (TRR) mRNA
used for treatment of neuropathy of hereditary transthyretin-mediated amyloidosisin
what are viruses?
replication particles with nucleic genomes
they must infect LIVING cells to proliferate
they can vary with regard to their structure and ability to INFECT DIFF HOSTS
how do viruses differ?
- host range = cell that is infected by virus is called host cell –> host range is number of species that a particular virus can infect
- structure = all viruses have NUCLEIC ACID GENOME (DNA or RNA) surrounded by a PROTEIN CAPSID
- some viruses have VIRAL ENVELOPE which is derived from plasma membrane of host cell and contains VIRAL SPIKE GLYCOPROTEINS
- genome composition = genome of virus can be DNA or RNA, can be ss or ds, and can carry a few of many genes
describe the viral reproductive cycles
series of steps that results in the production of new viruses, cycle occurs after a virus has infected a host cell, details of cycles vary from one type of virus to another but they all follow 5-6 basic steps
- attachment = virus attaches to surface of a host cell
- entry = virus or viral genome enters host cell
- integration = some but not all viruses integrate their genome into the genome host cell
- synthesis of viral components = viral proteins and RNA or RNA are made by host cell
- viral assembly = viral components assemble into virus particles
- release = viruses released from host cell
AEISAR
what does latency mean in viruses?
viruses may remain INACTIVE or LATENT, during which new viruses ARE NOT MADE
some bacteriophages are latent when they are in LYSOGENIC CYCLE
HIV usually remains latent for A LONG TIME after RNA genome is reverse transcribed into DNA and integrated into host chromosome
some viruses (like herpesviruses) can remain latent as EPISOMES = genetic element that can replicate independently of host genome
how does HIV work?
HIV causes AIDS, infects human T cells (important in immunity)
HIV has RNA genome, retrovirus
during cycle, HIV RNA is reverse transcribed into DNA and integrated into a host chromosome (may remain latent for a long time) –> after activation, new HIV particles are made by transcription of HIV and production of new HIV proteins that assemble at plasma membrane and bud from cell
HIV carries 9 genes, but some of them encode polyproteins that are cut into multiple proteins by proteolytic processing
explain the structure of HIV
has 2 copies of RNA genome that is surrounded by CAPSID –> capsid is surrounded by ENVELOPE that is derived from host cell plasma membrane and is studded with CIRAL SPIKE GLYCOPROTEINS
many of the HIV proteins are contained within the virus including REVERSE TRANSCRIPTASE and INTEGRASE (needed during early steps in viral reproductive cycle)
what is the genome of HIV?
9 genes of HIV can be divided into 5 categories
- gag = proteins used for VIRAL ASSEMBLY and CAPSID FORMATION, gene encodes polyprotein that is cleaved into 4 diff proteins (matrix protein, capsid protein, nucleocapsid protein, and p6’-role in release of virions from membranes of infected cells)
- pol = enzymes needed for VIRAL REPLICATION AND VIRAL ASSEMBLY, gene encodes a polyprotein that is cleaved into 3 enzymes (HIV protease, reverse transcriptase, and integrase)
- vif, vpu = proteins that promote INFECTIVITY and BUDDING
- vpr, rev, tat, nef = proteins with REGULATORY FUNCTIONS
- env = proteins that are part of VIRAL ENVELOPE –> gene encodes a polyprotein that is cleaved into 2 proteins (gp41 and gp120)
what allows HIV to infect host cells?
they can only infect host cells that have the required CD4 marker plus the coreceptor (CCR5 or CXCR4)
what is the HIV reproductive cycle?
spike glycoproteins on the HIV bind to receptors on the host cell membrane, the viral envelope fuses with the membrane, releasing the capsid and its contents
after HIV enters host cell, ssRNA is reverse transcribed into dsDNA by REVERSE TRANSCRIPTASE –> process begins when a host cell tRNA binds to the HIV RNA (tRNA acts as a primer) –> in series of steps, portions of viral RNA are reverse transcribed into DNA, viral DNA is used as templated to make complementary DNA strand to give dsDNA –> during this process, viral RNA is degraded by RNase H (component of reverse transcriptase) –> dsDNA is integrated into a random position in genome by HIV encoded integrase protein
when host cell receives signal to become active, provirus uses host RNA poly to create copies of HIV genome and shorter strands of mRNA which are translated –> assembly of virus particle takes place on INNER SURFACE of cell membrane (in macrophages and vacuoles)
HIV enzyme called protease cuts the long chains of HIV proteins into small proteins, the smaller HIV proteins come together with copies of HIV’s RNA genetic material and a new virus particle is assembled
gag precursor (Pr55gag) is major virion component, gets cleaved into 4 major proteins (matrix, capsid, nucleocapsid, and p6gag) and associates into virions spontaneously –> newly assembled virus buds out from host cell –> during budding, new virus gets covered by cell’s cells outer envelope (studded with glycoproteins to help virus bind to CD4 and coreceptors)
what are therapies for HIV?
viral life cycle have been targeted for antiviral therapy
combo therapy of use of multiple anit-HIV drugs like one protease inhibitor with 2 reverse transcriptase inhibitors
what is significant about +ve or -ve strand viruses?
+ve strand viruses have RNA genome that can be translated (mRNA like)
-ve RNA viruses CANNOT be translated
RNA synthesis has no…
proofreading mechanism
which influenza virus causes the most infections?
types A, B, and C but A causes the most infections
what kind of virus is the influenza virus?
orthomyxovirus
explain the components of the influenza virus
NEGATIVE (means cannot be translated), ssRNA
has 8 segmented genomes in A and B
3 proteins = M2, hemagglutinin, and neuraminidase in host cell-derived membrane on surface of virion
matrix protein M1 underneath, RNA segments covered in nucleoprotein
virus attaches to and multiples in the cells of the RESPIRATORY TRACT
describe the cycle of infection by influenza
- virus binds to the a respiratory epi cell by hemagglutinin spikes and fuses with the membrane
- virus is endocytosed into a vacuole and uncoated to release its 8 nucleocapsid segments into the cytoplasm
- nucleocapsid are transported into nucleus where the - sense RNA is transcribed into + sense strand that will be translated into viral proteins that make up the capsid and spikes
- (+) sense RNA is used to synthesize glycoprotein spikes inserted into host membrane
- release of mature virus occurs when viral parts gather at the cell membrane and are budded off with an envelope containing spike
- (+) sense RNA stands are used to synthesize new - sense RNA stands –> these are assembled into nucleocapsids and transported out of the nucleus to the cell membrane
why are influenza glycoproteins important?
- hemagglutinin (H) = 18 diff subtypes, most IMPORTANT VIRULENCE FACTOR, binds to host cells
- neuraminidase (N) = 11 subtypes, HYDROLYZES MUCUS and ASSISTS VIRAL BUDDING and RELEASE
H and N are used to classify the influenza viruses (H1N1)
both glycoproteins frequently undergo genetic changes, producing new variant viruses
what is antigenic DRIFT?
viral RNA poly is ERROR-PRONE, resulting in constant MUTATIONS and results in AA subs in the viral proteins
what is antigenic SHIFT?
one of the genes or RNA stands is sub with a strand from ANOTHER influenza virus of a different animal host by REASSORTMENT
what is needed for influenza to be infective?
it needs all 8 RNA segments to be present to be an infectious virus
what happens to the RNA segs when a cell is infected by 2 different influenzas?
RNA replication will produce a mix of the 8 RNA segs from the avian virus and from the human virus
the packaged virus will contain a mix of human and avian RNA segs
this is known as REASSORTMENT and produced new combos of HA and NA (antigenic shift)
what are the 3 reservoirs of influenza viruses?
aquatic birds
pigs
humans
when reassortment occurs, the new virus will have to be very diff from all existing viruses (worldwide epidemics may be a consequence since the entire pop is susceptible to the virus)
explain the components of coronaviruses
large RNA viruses with spaced spikes on envelopes
+ve strand RNA (can be translated into mRNA), non -segmented, large RNA genomes
common in domesticated animals
7 coronaviruses have been id as causes of human disease, ex: MERS, SARS, SARS-2CoV
virus has spikes, nucleocapsids on its RNA viral genome which is enclosed in an envelope
what is the reproductive cycle of coronaviruses?
- binds to host cell receptor ACE-2
- once endocytosed, its viral genome is released
- the RNA genome is replicated
- the viral RNA is transcribed
- translation of the viral proteins using host machinery
- assembly of the viral particle
- formation of the mature virion
- exocytosis of the new virion
what components of the coronoviruses are targets for vaccine development?
spike protein is a common target for anntibody and vaccine development
another strategy = inject indvs with DNA/RNA encoding viral proteins
- live attenuated
- whole inactivated
- RNA
- DNA
- recombo subunits
- recombo viral vectors
what is a mutation?
a heritable change in gene material
mutations provide allelic variations
mutations are more likely harmful than beneficial
what is a point mutation?
change in a single base pair/adjacent base pairs
it can involve a base substitution
this includes transitions and transversions
mutations may also involve addition or deletions of short DNA seqs
what is a transition?
a change of a pyrimidine (C, T) to another pyrimidine or a purine (A, G) to another purine
transitions are more common than transversions
what is a transversion?
a change of pyrimidine to a purine or vice versa
what are silent mutations?
base subs that DO NOT ALTER the AA seq of the polypep (due to degeneracy of genetic code)
what are missense mutations?
base subs in which AA change does occur
ex: sickle cell anemia from glutamic acid to valine
if the sub AA has no detectable effect on protein function, the mutation is NEUTRAL
this can occur if new AA has similar chem to the AA it replaced
what are nonsense mutations?
base subs that change a normal codon to a STOP codon (can result in shorter proteins)
what are frameshift mutations?
addition or deletion of number of nucleotides that is NOT DIVISIBLE BY 3
shifts reading rame so that translation of mRNA resutls in completely diff AA seq downstream of mutation
what is the frameshift mutation of HIV?
caused by a 32 bp deletion in CCR5 gene, results in an inactive (undetectable) protein and near complete resistance to HIV-2 infection (present in 13% of N. Europeans)
what are the effects of gene mutations outside of the coding seqs?
they can still affect phenotype
mutations in the core promoter can change levels of gene expression
- up promoter mutations = increase expression
- down promoter mutations = decrease expression
- regulatory element/operator site = may disrupt ability of gene to be properly regulated
- 5’UTR/3’UTR = may alter ability of mRNA to be translated, may alter mRNA stability
- splice recognition seq = may alter ability of pre-mRNA to be properly spliced
what are forward mutations?
changes the wt genotype into some new variation
what are reverse mutations?
changes mutant allele back to wt
also called reversion
what are deleterious mutations?
decrease the chances of survival
most extreme = lethal mutations
what are beneficial mutations?
enhance survival or reproductive success of an organism
environment can affect whether a given mutation is deleterious or beneficial
what are conditional mutations?
they affect phenotype only under a defined set of conditions
ex: temp-sensitive mutation
what are the 2 animal cell types?
germ-line cells = cells that give rise to gametes
somatic cells = all other cells
what are germ line mutations?
occur directly in sperm or egg or in one of their precursor cells
can be passed to next gen where mutation will be found in whole body
what are somatic mutations?
those that occur directly in a body cell or in one of its precursor cells, NOT PASSED ONTO NEXT GEN
result in patches of affected area, size of patch will depend on timing of mutation (earlier mutation = larger patch)
indv with somatic regions that are genotypically diff is called GENETIC MOSAIC
when do mutations arise?
arise spontaneously and continuously during culture, early arising mutants will give rise to LOTS OF MUTANT COLONIES
late arising mutants will give rise to FEW RESISTANT COLONIES
number of resistant colonies in cultures should FLUCTUATE
what were the 2 possibilities of mutations for T1-resistant mutants?
- mutants arise spontaneously and continuously aka spontaneous mutation (if T1-resistant mutants are are already in culture before T1 exposure, then there will be variation and fluctuation which supports the hypothesis that resistance to T1 arises from pre-existing random mutations)
- T1-resistance mutants arise ONLY AFTER exposure to T1 (in this case, see no fluctuation, any variation among the cultures and aliquots from same large culture should be the SAME) aka induced mutation
mutations were continuous and random, they were pre-existing and selected by environmental agent
what is the physiological adaptation theory?
predicts that the number of tonr (T one resistance) bacteria is very low unless there is a selection, when the numbers created increase
what is the random mutation theory?
predicts that the number of tonr bacteria will fluctuate in diff bacterial pops and will occur without selection
explain the process of the random mutation theory in relation to resistance of E. coli to infection by bacteriophage T1
- place indv bacterial cells onto growth media
- incubate overnight to allow formation of bacterial colonies = master plate
- press velvet cloth onto master plate and then lift gently to obtain a replica of each bacterial colony –> press replica onto 2 secondary plates that contain T1 phage –> incubate overnight to allow growth of mutant cells
resistant cells were in the same location on both plates
mutations had randomly occurred in absence of selection by T1
became observable with selection for T1 resistance
supports random mutation theory
what are spontaneous mutations?
result from abnormalities in cell/biological processes
ex: errors in DNA replication, DNA damage from normal cell processes
underlying cause originates with the cell
what are induced mutations?
caused by environmental agents
agents that are known to alter DNA structure are termed mutagens (can be chemical or physical agents
what are the 4 types of chemical changes that can cause spontaneous mutations?
- depurination
- deamination
- tautomeric shift
- oxidative stress
explain depurination
removal of a purine (A or G) from DNA forming an APURINIC SITE
apurinic site can be repaired, however, if the repair system fails then a mutation may result during subsequent rounds of DNA replication (poly will add a random base) –> 3/4 bases are incorrect nucleotide (75% chance of mutation)
explain deamination of cytosine
removal of an amino group from the cytosine base (other bases are not readily deaminated)
DNA repair enzymes can recognize U as an inappropriate base in DNA and remove it
however if the repair system fails, G-C to A-T mutat will result during subsequent rounds of DNA replication
G-C –> G-U –> A-U –> A-T (NOT T-A)
explain deamination of 5-methylcytosine
5-methylcytosine typically occurs at cytosine in CpG di-nt in eukaryotic DNA
5-methylcytosine can be deaminated into THYMINE, a normal constituent of DNA
repair enzymes CANNOT determine which of the 2 bases on the 2 DNA strands is the correct base –> for this reason, methylated cytosine bases tend to create hot spots for mutation
what is tautomeric shift?
spontaneous isomerization of a nitrogen base to an alternative hydrogen-bonding form
temporary change in a base structure
common, stable form of THYMINE and GUANINE is KETO FORM (RARELY T and G convert to enol form)
common stable form of ADENINE and CYTOSINE is the AMINO FORM (RARELY, A and C can convert to an IMINO FORM)
rare tautomers can engage in erroneous base pairing
what can tautomeric shifts cause?
tautomeric shifts and DNA replication can cause mutation
to cause mutation, tautomeric shift must occur IMMEDIATELY PRIOR TO REPLICATION
T-A –> T-G–> C-G
what DNA damage does ROS cause?
oxidative DNA damage
guanine can be converted to 7,8-dihydro-8-oxoguanine
- pairs with adenine during replication (8-oxyG-A)
- causes GC base pair –> G-A–> TA base pair
2 steps: chemical alteration of DNA base –> altered pairing by this altered base at next round of DNA replication
what are tri-nt repeats?
many human genetic diseases are caused by an unusual form of mutation called tri-nt repeat expansion (TNRE)
certain regions of the chromosome contain tri-nt seqs repeated in tandem
–> these repeats are prone to EXPANSION/CONTRACTION in the genome
in norm indvs, these seqs are transmitted from parent to offspring intact w/o extra copies but in people with TNRE disorders, length of tri-nt repeat increased above critical size (when threshold number of repeats has been exceeded, disease symptoms occur
what occurs during replication slippage at repeated seqs?
insertion = reannealing (of new stand) occurs UPSTREAM of original position
deletion = reannealing occurs DOWNSTREAM of original position
what is the tri-nt repeat disease?
FRAGILE X SYNDROME = most common form of inherited mental impairment
one end of X-chromosome looks like it’s hanging by a thread –> disease is transcribed but NOT TRANSLATED
syndrome caused by INCREASE in number of CGG repeats in FMR-1 gene
mutated (expanded CGG) region is in part of gene that is transcribed but not translated (5’ UTR)
DOMINANT, X-LINKED
unaffected parents can have affected offspring
what is the threshold for tri-nt repeat disease?
200 repeats
how does tri-nt repeats affect the FMR-1 gene?
silencing of the FMR-1 gene associated with CpG methylation and shift in epigenetic marks of chromatin from euchromatic state to heterochromatic state –> results in NO TRANSCRIPTION of gene
increased CGG repeats turn off transcription
what repeat number will cause a defect in the replicated seq?
if repeat number is greater than 50, correct seq cannot be replicated by replication machinery –> expanded
what do tri-nt disease lead to?
many tri-nt diseases lead to increases in repeats of a SINGLE AA (repeats in exons, like CAG, resulting in polyglutamine tract in the protein) –> this causes proteins to aggregate with each other which is correlated with progression of disease (like Huntington’s)
why are we concerned about mutagens?
agents that alter the structure of DNA thereby cause mutations are called mutagens
we are concerned about mutagens because:
1. they are often involved in development of HUMAN CANCERS
2. they can cause GENE MUTATIONS that may have harmful effects in future generations
mutagenic agents are usually classified as chem or physical mutagens
how do mutagens alter DNA structure?
- base modifers = covalently modify base structure, some may disrupt pairing by AKLYLATING bases –> base sub mutation
- intercalating agents = directly interfere with REPLICATION PROCESS (deletions, insertions, frameshift mutations)
- base analogs = resemble DNA bases, incorporate into DNA and disrupt structure, some tautomerize at high rate –> base sub mutation
physical mutagens include radiation (x-rays, gamma rays, ionizing radiation, UV light)
what are base modifiers?
covalently modify the structure of a nt (altered bases are now mispairs)
ex: NITROUS ACID replaces amino groups with keto groups, this can change cytosine to uracil and adenine to hypoxanthine (modified bases DO NOT PAIR with appropriate nt in DAUGHTER strand during DNA replication)
some chemical mutagens disrupt the appropriate pairing between nucleotides by alkylating bases within the DNA
ex: nitrogen mustards (NITROSGUANINE) and ethyl methanesulfonate (EMS)
alkylating agents EMS (adds ethyl) and NG (adds met+) to about all bases but mostly G–>O-6-alkylguanine (which mispairs with thymine)
causes G:C –> A:T transitions
(know that these chemicals are base modifying agents and cause base subs)
what are intercalating agents?
contain flat, planar structures that intercalate themselves into double helix –> distorts helical structure
when DNA containing these mutagens is replicated, daughter strands may contain single-nt additions/deletions resulting in frameshifts
ex: PROFLAVIN, ACRIDINE ORANGE, ICR-191 (know that these chemicals are intercalating agents and cause FRAMESHIFTS)
what are base analogs?
resemble normal bases
become incorporated into daughter strands during DNA replication
ex: 5-bromouracil is a THYMINE analog –> can be incorporated into DNA instead of thymine and results in tautomeric shift that pairs with GUANINE
A-T –> A-BrU –> G–BrU –> G-C
base pairing of 5-BrU with adenine or guanine
know that these chemicals are base analogs and cause BASE SUBS and knows steps involved in generating base changes
how does 5BU cause a mutation?
A:5BU –> A:T or G:5BU –> G:C or G:5BU
what does ionizing radiation cause?
includes x-rays and gamma rays
has short wavelength and high energy, can penetrate deeply into bio samples
has 2 effects on DNA:
1. (indirect) generates ROS, react with DNA –> mutation
2. (direct) break DNA backbone, to give ss or ds breaks, or break glycosidic bond –> AP sites
can cause:
- base deletions
- oxidized bases
- single nicks in DNA strands
- cross-linking
- chromosomal breaks
what does nonionizing radiation cause?
includes UV light
has less energy
cannot penetrate deeply into bio molecules
causes formation of CROSS-LINKED THYMINE or pyrimidine dimers (joins adjacent Ts on same strand)
damage prevents base pairing, blocks replication
thymine dimers may cause mutations when DNA strand is replicated
what does aflatoxin B1 form?
aflatoxin, potent mutagen and carcinogen
modifies guanine and adds bulky adduct
causes G–>T TRANSVERSIONS
how is the Ames test used?
forward mutation (wt–>mut, or his+ –> his-)
detect his- by growing all bacterial on MM + histidine then testing colonies on MM alone –> those that grow on MM+ His but not MM alone are his- (because his- needs his to grow)
this method takes too long because there’s thousands and millions of colonies
reverse mutation (mut –> wt, or his- –> his+)
detect these by planting the culture of his- bacteria on MM with no his
only his+ revertants will grow on MM
can screen 10^9 cells on a single plate and detect very low freq of reversion very easily
what is the purpose of the Ames test?
test uses strains of S. typhimurim that CANNOT synthesize the AA his
each has a point mutation (his-) in a gene involved in his biosynthesis –> strain is unable to grow on MM unless his is supplied or reversion mutation occurs, restoring wt seq and ability to synthesize his
Ames test monitors rate at which this reversion mutation occurs
what is the Ames test used for?
used to screen possible carcinogen and mutagens
salmonella his auxotroph (requires his to grow) are mixed with rat liver enzymes and plated on media lacking his
rat liver extract provides a mix of enzymes that may activate a mutagen
live enzymes are required to detect mutagens that are converted to carcinogenic forms by liver
test chemical is then added to medium
control plates show only a small # of revertants (bacteria cells growing without his)
plates inoculated with mutagens/procarcinogens show a larger # of revertants
what is the Ames test of aflatoxin B1 mutagenicity?
different Ames strains (TA strains) have diff mutations that can report on distinct mutagenic events
some his- mutations are frameshifts, can only be reverted by mutagens that induce frameshifts
TA100 = base subs
TA1538 = frameshift (-1)
what are the steps of DNA repair?
- an irregularity in DNA structure is detected
- abnormal DNA is removed
- normal DNA is resynthesized
what are some enzymes that can reverse covalent mods of nts?
PHOTOLYASE can repair thymine dimers –> splits dimers, restoring DNA to its original condition, uses ENERGY of VISIBLE LIGHT for photoreactivation, will not repair dimers in dark
ALKYLTRANSFERASE repairs alkylated bases –> transfers methyl or ethyl group from base to cysteine side chain with alkyltransferase protein –> will permanently inactivate alkyltransferase
what is the function of excision repair?
remove damage (base or DNA backbone)
ss nick/gap provides 3’ OH for DNA pol I initiation
DNA ligase seals nick
REMOVE AND REPLACE mechanisms
what are the types of excision repairs?
- base excision repair = cut out base, then cut next to apurinic/aplyrimidinic site and let DNA pol I repair
- nt excision repair = cut out seg of DNA around a damaged base
explain base excision repair (BER)
involves enzymes known as DNA N-glycosylases –> these enzymes can recognize an abnormal base and cleave the bond between it and sugar in the DNA via AP endonuclease –> AP site
depending on species, this repair system can eliminate abnormal bases like URACIL, 3-METHYLADENINE, and 7-METHYLGUANINE
AP endonuclease cleaves backbone upstream of AP site
DNA poly I fills in ss gap, using remaining undamaged DNA strand as template
ligase seals the nick in backbone
explain nucleotide excision repair
BER CANNOT repair bases with bulky adducts that distort helix or remove multiple, adjacent damaged bases –> unless repaired, these can block TRANSCRIPTION and DNA REPLICATION
this is why NER is important –> this type of system can reapir many types of DNA damage including:
- thymine dimers (if not repaired by photolyase) and chemically modified bases, missing bases, and some types of crosslinks
NER is found in all eukaryotes and bacteria
in E. coli, NER system requires 4 key proteins –> UvrA, UvrB, UvrC, UvrD (involved in Ultraviolet light Repair of pyrimidines) –> they will recognize and remove a short seg of damaged DNA, DNA poly and ligase will finish the job
UvrA/UvrB complex tracks along DNA in search of damaged DNA –> after damage is detected, UvrA is released and UvrC binds –> UvrC makes cuts on both sides of thymine dimer –> UvrD (helicase) removes damaged region, UvrB and UvrC are released –> DNA poly fills in gap and DNA ligase seals the region
what diseases are involved in defects in genes of NER?
xeroderma pigmentosum (XP), Cockayne syndrome (CS), and PIBIDS
common characteristic of all 3 syndromes = increase sensitivity to sunlight
XP can be caused by defects in 7 diff NER genes
what is the mismatch repair system?
recognize and correct a base pair mismatch
structure of DNA double helix obeys AT/GC rule but during DNA replication, an incorrect base may be added to growing stand by mistake
DNA poly have 3’-5’ proofreading ability that can detect base mismatches and fix them
if proofreading fails, mismatch repair system comes to rescue
mismatch repair systems are found in all species
important aspect of these systems is that they are specific to newly made strand
explain how mismatches are detected
using the proteins MutL, MutH, and MutS –> they also direct mismatch removal from newly made strand (proteins named Mut because their absence leads to higher mutation rate than norm)
what is important about MutH?
if can distinguish between parental and daughter strand
prior to replication, both strands are methylated –> immediately after replication, parental strand is methylated while daughter is NOT (hemimethylated)
in E. coli, A of GATC seqs is methylated
explain the process of mismatch repair
MutS protein slides along DNA and finds mismatch
MutL is recruited
MutS/MutL complex binds to MutH, which is already bound to hemimethylated seq
MutH cuts unmethylated stand with wrong base
region including mismatched base removed, leaving gapped DNA
repair synthesis resynthesizes removed ssDNA
how are double-strand breaks in DNA repaired/
by recombo
repair pathways use undamaged strand as template in resynthesis reaction
DNA ds breaks are VERY DANGEROUS –> cause breakage of chromosomes into pieces –> breaks are caused by IONIZING RADIATION and CHEMICAL MUTAGENS (also ROS) –> breaks can cause chromosomal rearrangements and deficiencies
may be repaired by 2 mechanisms:
- HOMOLOGOUS RECOMBO REPAIR (HRR)
- NONHOMOLOGOUS END JOINING (NHEJ)
if DSB in dividing cell with sister chromatid available –> break can be repaired using undamaged chromatid as template –> DSB is processed by short digestion of DNA strands (used for strand exchange)
explain repair of DSB by HRR
unbroken stands are used as templates to synthesize DNA
strands are then broken and rejoined in way that produces separate chromatids
because sister chromatids are genetically identical, homologous recombo can be ERROR-FREE repair mech
has ss projections that will pair with template
explain repair of DSB by NHEJ
broken ends are recognized by end binding proteins KU70/KU80
proteins form a cross bridge, other proteins are recruited, ends trimmed to generate 5’P and 3’OH –> this processing may result in deletion of small region
ends joined by ligase
INHERENTLY ERROR-PRONE/MUTAGENIC
how is damaged DNA replicated?
by translesion DNA pols
but before this –> replicative DNA pols like DNA pol III are sensitive to distortions in DNA –> unable to replicate through DNA lesions, this type of replication requires specialized DNA pols, these pols are induced as part of DNA damage response (SOS response) and will insert any nt into damaged region
TLS pols contain active site with loose, flexible pocket –> can accomodate abberant structures in template strand –> neg. consequence of TLS pols = low fidelity (mutation rate is 10-2 to 10-3, higher than 10-8 of replicative pol)
when replicative DNA pol encounters damaged region, it is swapped for TLS poly which can tolerate template bases with bulky adducts –> inserts a few bases then falls off, pol III is reattached –> region is replicated but with ERROR-PRONE REPLICATION
what is the purpose of dosage compensation?
to offset differences in number of active sex chromosomes and ensure equal gene expression in both
X-chromosome inactivation
how does dosage compensation occur?
occurs by chromosome condensation
Barr body is a highly condensed, inactivated X chromosome (typically only seen in females)
what is the mechanism of X-chromosome inactivation (dosage compensation)?
female mouse has inherited 2 X chromosomes (one from mother that carries allele conferring white fur Xb and one from father that carries allele conferring black fur XB)
random x-chromosome inactivation occurs EARLY in development
in some cells, X w/ b (white) allele is activated so only some XB allele is active –> these cells and their descendants only express black fur –> opp is true for cells that inactivated XB (descendants only express white fur)
during x-chromosome inactivation, DNA becomes highly compacted, most genes on inactivated X CANNOT be expressed, inactivated X is maintained in an inactive state through replication and cell division (x-chromosome inactivation pattern is passed along to all future somatic cells)
ex: calico cat with black and orange fur (they always FEMALE)
what is significant about X chromosomes?
mammalian can count their x chromosomes and allow only one of them to remain active –> additional x chromosomes are converted to Barr bodies
XX = 1 barr body
XY = 0 barr body
X0 = 0 barr bodies
XXX = 2 barr bodies
XXY = 1 barr body
what is gene cloning?
technique of isolating and making many IDENTICAL copies of a gene
what are the two kinds of DNA molecules in cloning experiments?
- CHROMOSOMAL DNA (the ‘insert’) = serve as source of DNA segment of interest –> to prepare chromosomal DNA –> obtain cell tissue from organism of interest, break open cells, extract and purify DNA using variety of biochem techniques
- VECTOR DNA = usually bacterial plasmid, serve as CARRIER FOR DNA seg that is cloned, can replicate independently of host chromosomal DNA
why is vector DNA important in cloning?
cell that harbors vector is called HOST CELL (usually E. coli)
when vector is replicated inside host cell, DNA that it carries is also replicated
the seq of ORIGIN of REPLICATION determines whether a vector can replicate in a particular host cell
what 2 sources were vectors originally derived from?
- plasmids = many naturally occurring plasmids have SELECTABLE MARKERS, typically genes conferring ANTIBIOTIC RESISTANCE TO HOST CELL
- viruses/phages
explain the process of DNA cloning
vector is cleaved with restriction endonuclease and DNA frag from organism of interested is added to the cleaved portion by ligase –> recombo vector DNA is introduced into host cell –> propagation produces many copies of recombo DNA
why are restriction enzymes important?
insertion of chromosomal DNA into a vector requires cutting and joining of DNA frags –> these enzymes are restriction endonucleases or restriction enzymes
protect bacterial cells from invasion by foreign DNA (particularly that of bacteriophage) –> these enzymes bind to specific DNA seqs and then cleave the DNA at 2 defined locations, one on each strand
they always have accompanying methylase which methylates the same seq, prevents cutting
bacteria have methylase and RE, so its own DNA is immune from cutting by RE so methylated DNA IS NOT CUT but UNMETHYLATED IS CUT
where do restriction enzymes cut DNA?
at specific sites called palindromic seqs
the seq is identical when read in opposite direction in complementary strand
BamHI = 5’ GGATCC 3’ or Sau3AI = 5’ GATC 5’
ex: 5’ AGCT 3’ or 5’ AAGCTT 3’
3’ TCGA 5’ 3’ TTCGAA 5’
what is another function of restriction enzymes?
some will digest DNA into fragments with STICKY ENDS = short, ss regions of DNA that can base-pair with another piece of DNA with complementary ss seq
some will generate blunt ends –> enzyme NaeI cuts in middle of recognition seq (5’ GCCGGC 3’)
sugar-phosphate backbone of DNA molecules with stick yor blunt ends can be covalently linked together by ligase
what does the plasmid vector pUC18 carry?
ampR –> confers ANTIBIOTIC RESISTANCE to host cell (id cells that have taken up vector)
lacZ –> encodes beta-galactosidase (provides means by which bacteria have picked up cloned gene can be id)
- origin of replication = allows plasmid to replicate independently of chromosome of bacterial host cell
what is pBR322 resistant to?
amp and tet
what are the steps in gene cloning?
human chromosomal DNA would generate thousands of frags, typically one frag inserts into plasmic vector at any one time (cut plasmid DNA and human DNA with same restriction enzyme, mis, add ligase) and becomes RECOMBO PLASMID
transform E. coli cells with ligation mix, select on amp (or whatever antibiotic plasmid confers resistance to), cells that are able to take up DNA are called COMPETENT CELLS, transformation refers to uptake of plasmid vectors by a bacterial cell, single competent bacterial cell takes up single plasmid molecules and goes on to form a colony on an amp agar plate
all bacterial colonies growing on amp plate must have picked up the vector and its ampR gene
how do you distinguish between colonies in gene cloning?
colonies may have recircularized plasmid (no insert DNA) or have recombo plasmid, carrying inserted DNA –> this is where lacZ gene comes into play
in hybrid vector, chromosomal DNA inserts into lacZ gene, thereby disrupting it (lacZ+ –> lacZ-) –> recirculized vector has FUNCTIONAL lacZ+ gene
how is the functionality of lacZ gene determined?
2 relevant compounds
- IPTG which induces lacZ gene expression
- X-Gal = colorless compound that is cleaved by beta-galactosidase into BLUE DYE (color of bacterial colonies will depend on whether or not beta-galactosidase enzyme is function)
how is lacZ used to id recombo plasmids?
in this experiment, when plated on media w/ amp, IPTG, and X-gal –> colonies with RECIRCULARIZED (lacZ+) vectors form BLUE colonies (bad because not recombo) while those with that are hybrid (lacZ-) vectors form white colonies (good because they are recombo plasmids)
what are the 2 ways in which cloned genes are amplified?
- vector gets replicated by host cell many times (this will generate a lot of copies per cell)
- bacterial cell divide about every 20 mins(this will generate pop of many millions of cells overnight)
what is cDNA from from mRNA?
using reverse transcriptase
clone of eukaryotic gene made from chromosomal DNA will contains introns, exons, and control regions –> you want to clone a of eukaryotic gene that contains full ORF with NO INTRONS
enzyme reverse transcriptase is used to make DNA from RNA –> uses RNA as templated to make complementary DNA (cDNA)
- add poly-dT primer that binds to the polyA tail of mRNA
- add reverse transcriptase + dNTPS to synthesize cDNA
- add RNase H to cut up RNA and generate RNA primers
- add DNA pol I and ligase to synthesize 2nd DNA strand
why is cDNA cloning useful?
advantage of cDNA is that it lacks introns
if you want to express human gene in C. color and recover recombo protein, recombo plasmid will need to:
- be a cDNA clone (exons/coding seqs only, no introns)
- have an E. coli promoter
- have an E. coli ribosome binding site
- eukaryotic specific post-translational mods will NOT occurs
what is a DNA library?
treating chromosomal DNA with REs can generate tens of thousands of frags
after ligation, there is a collection of recombo vectors, with each vector containing a certain frag
if the starting material was chromosomal DNA = genomic library
if starting material was cDNA, library is called cDNA library
what is the function of PCR?
it can copy a specific sequence of DNA without aid of vectors and host cells (must know enough about the gene of interest to have the sequence of 2 short primers)
researched much have prior knowledge about the seq if the template DNA to structure the synthetic primers –> specific DNA seg can be amplified from a complex mix of other seqs
- you can amplify one gene out of an entire genome/mixture of diff genomes
- PCR can be used to amplify very small samples (like in forensics) or retrieved from ancient bio specimens
- DNA degrades over time
- mtDNA has multiple copies/cell, so there’s a better chance of recovering it from ancient samples
how do you amplify DNA using PCR?
to amplify DNA by PCR:
1. template DNA (contains region that needs to be amplifed)
2. oligo-nt primers (complementtary seqs at ends of DNA frag to be amplified, these are synthetic and about 15-20 nts long)
3. deoxynucleoside triphosphates (dNTPs) –> provide precursors for DNA synthesis
4. Taq poly = DNA poly isolated from Taq, THERMOSTABLE enzyme is necessary because PCR involves heating steps that inactivate most other DNA pols
what are the steps in the PCR cycle?
- denaturation
- primer annealing
- primer extension
KNOW THIS ORDER - add oligo-nt primers
- heat to separate strands
- cool and let primers anneal (55-65 C)
- heat to allow DNA synthesis (Taq works)
repeat steps 2 and 3
what happens in repeated cycles in PCR?
carried out in a thermocycler
all ingredients placed into one tube –> experimenter sets temp range and number of cycles (sequential process of denaturing-annealing-synthesis is repeated for 25-35 cycles)
since very little starting template DNA is needed, PCR is highly sensitive
how are pathogens detected by PCR?
small sample size, no need to culture organism/virus
look for amplification of a specific DNA frag characteristic or unqiue to a specific org (use PCR primers that will anneal to DNA only from specific organisms)
what is the function of reverse transcriptase PCR?
used to detect and quantitate amount of RNA in living cells
RNA is isolated from a sample, mixed with RT and a primer that will anneal to the 3’ end of the RNA (gene-specific primer)
generates a ss cDNA which can be used as template DNA in conventional PCR
first PCR cycle will convert ss cDNA to dsDNA
what is real time PCR or quantitiative qPCR?
used to quantitate the amount of a specific gene or mRNA in a sample
real time PCR is carried out in a thermocycler that can measure changes in fluorescence emitted by detector molecules in PCR reaction mix –> can be used to quantify amount of specific piece of DNA or RNA present in sample
fluorescence is detected by thermocycler and will increase in proportion to amount of PCR product produced
PCR product in tern is proportional to amounnt of DNA or RNA present in original sample
this is being used to detect Covid-19 (viral RNA –> cDNA –> amplied in qPCR reaction)
greater amount = greater fluorescence
explain gene editing
analysis of mutations can provide important info about normal genetic processes
mutations can arise spontaneously, be induced by mutagens, or created with rDNA
researchers have developed techniques to
- make mutations within cloned DNA
- introduce these mutant genes into org, replacing the WT copy of gene
gene editing = experimentally altering a gene seq
how is a knockout mutation made?
by replacing WT gene with a drug resistance gene
recombo is facilitated by homo regions flanking the drug-resistance cassette
replacing a WT gene iwth a deleted one, carrying a drug R marker (what you would like to happen) but what actually happens is that the introduced DNA frag carrying drug R integrates at a non-homologous position
how is a knockout mutation made in mice?
make alterations in mouse embryonic stem cells
introduce these stem cells into blastocysts
implant chimeric blastocyst in pseudo-preg mouse
look for offspring who’s germline has be modified
process is caused by non-homo, ectopic recombo
homo recomb is very inefficient, so you can’t use single cell zygotes which is why you have to resort to stem cells
what are solutions to non-homo recombo problem in generating mouse knockout?
freq of homo recombo increased hugle if you can generate a DSB in/next to site where you want recombo to occur
DSBs are LETHAL and must be repaired
- repair by recombo with homo DNA, injected into cell at same time DSB occurs
- if no homo DNA around, repair by berror-prone non-homo end joining (NHEJ) –> generates insertions/deletions that knockout gene
what are the repair mechanisms for DSBs?
NHEJ = correction leads to constitutive KO
HRR correction based on provided template creates KI (knock in)
CRISPR/Cas9
explain the CRISPR/Cas 9 system
virus invades bacterial cell, new spacer is derived from virus and integrated into CRISPR seq, CRISPR RNA is formed, CRISPR RNA guides molecular machinery to target and destroy viral genome
RNA produced is complementary to invading phage/plasmid, crDNA base pairs to invading DNA (this is how specific DNA is targeted
2nd RNA, tracrRNA also NEEDED (functional RNA) –> Cas9 recruited, cleavage at site of DNA-RNA base pairing –> crRNA binds next to an NGG PAm seq –> incoming DNA destroyed by dsDNA cleavage
Cas9 makes guide RNA that is complementary to where you want to cut (as long as NGG PAM seq is present)
what can CRISPR/Cas9 do to DNA?
it can make DSBs at specific seqs in genome
these breaks have to be repaired
if you want to INACTIVATE gene –> can rely on error-prone NHEJ to repair break, introduces deletion/insertion (indels, usually ABOLISH GENE FUNCTION leading to gene KO)
if you want to INTRODUCE a specific CHANGE in a gene –> while gRNA/Cas is injected, you also deliver homo ‘repair’ donor DNA molecule –> DNA can be used to DSB by homo recombo (if DNA carries mutation then mutation will be recombined into repaired chromosome –> produces recombo chromosome with changes you want (KI mutations)
CRISPR/Cas9 will continue to cleave perfectly repaired chromosomes –> indel generated by error-prone NHEJ can NO LONGER be cleaved by CRISPR/Cas9 (this version of CRISPR/Cas9 can ONLY generate gene KOs)
what is the dideoxy method of DNA sequencing?
during replication, DNA poly connects adjacent deoxy-nts by covalently link 5’-P of one to 3’ OH of another
nt w/o 3’ OH can also be sunthesized = dideoxyribonucleotides of ddNTPS (cannot form bonds)
if ddNTP is added to a growing DNA strand, strand can no longer grow –> leads to chain termination
the 3’ OH group necessary for formation of phosphodiester bond is missing in ddNTPs –> chain terminates at ddG
what is manual DNA sequencing?
if you have a mix of dATP and ddATP, some chains will be terminated at EVERY T in template, others will continue to be elongated
length of these dd-A terminated chains correspond to position of Ts in template
prior to DNA sequencing, DNA must be obtained in large amounts (PCR or cloning) –> target DNA can be cloned into plasmid at site adjacent to primer annealing site –> target DNA is heat denatured into ss –> heat denaturation step provides ss DNA template for modified replication rxn in Sanger DNA sequencing
what happens when ddnt is incorporated in a seq?
the A reaction mix contains template primer, DNA poly, dGTP, dCTP, dTTP, dATP, and ddATP
at each T in template, either dATP or ddATP can be incorporated –> if ddA is incorporated, synthesis ends but if dA is incorporated, synthesis continues
partial termination at each T on template means that some nt chains are terminated, others continue to be elongated
the lengths of the nt chains ending in ddATP tells us where each T was in the template, primer is labeled with radioactive 32P so all chains are radioactive too, these chains are resolved on gel and visualized by autoradiography –> you read the seq of synthesized strand (5’ to 3’), BOTTOM TO TOP (smaller to larger)
T is RED every time ddTTP incorporated opposite A in template, C is PURPLE even time ddCTP incorporated opposite a G in template
what do the colors in Sanger sequencing mean?
the seq of diff colors corresponds to seq of bases in synthesized DNA
what is shotgun sequencing?
method where small DNA frags are randomly generated from larger pieces and sequenced
- most efficient and inexpensive way to seq genomes
- this method does not require extensive mapping, but the same region may be sequenced multiple times
regions of overlap are used to map the frags, relative to one another
explain the process of genome sequencing
- purify DNA from strains of bacteria –> involves breaking cells open by adding phenol and chloroform, most protein and lipid components go into pheno-chloroform phase –> DNA remains in aqueous phase, which is removed and used in step 2
- sonicate (using sound waves) DNA to break it into small frags
- clone DNA frags into vectors, producing a DNA library
- subject many clones to the procedure of ddDNA sequencing
- use tools of bioinformatics to id various types of genes in genome
what is the Human Genome Project?
- to obtain a genetic linkage map of human genome
- to obtain a physical map of human genome
- to obtain the DNA seq of entire human genome
- to develop technology for management of human genome info
what is high-throughput sequencing?
rapidly sequencing large amounts of DNA
possible because of:
- AUTOMATED SEQUENCING with fluro detector
- parallel sequencing = simultaneously perform many sequencing runs in multiple, gel-filled capillary tubes
- next gen sequencing technologies (NGS) = ability to process thousands or millions of seq reads in parallel
what is pyrosequencing?
one kind of NGS
DNA is fragmented
mix of DNA frags with adaptors called sample library
frags are attached to beads PCR amplified
sequencing reagents flow over beads in wells of plate: dG, dA, dT, dC
sequencing by synthesis (SBS) –> PPi is released when nt is added, ATP sulfurylase uses it to make ATP, ATP is used by luciferase to break down luciferin, rxn gives off light detected by a camera in the sequencing machine
- isolate genomic DNA and break into frags
- covalently attach oligonucleotide adaptors to 5’ and 3’ ends of DNA
- denature DNA into ss and attach beads via red adaptors (only one strand is attached to a bead)
- emulsify the beads so there is only one bead per droplet, droplets also ocntain PCR reagents that amplify the DNA
why is an ORF important?
it is a nt seq that does not contain any internal stop codons (just one at the end)
- in bacteria, long ORFs are contained in chromosomal gene seqs
- in eukaryotes, chromosomal coding seqs may be interrupted by introns
- in DNA seq, reading of codons could begin with 1st, 2nd, or 3rd nts (called reading frame 1, 2, 3)
since there are 2 strands of DNA, in any sdDNA frag, there can be 6 reading frames
what is BLAST?
it tells you if a ORF encodes a protein similar to a protein characterized and identified in a diff organism
basic local alignment search tool
searches in databases for AA or nt seq similarity, for whole gene or motifs
‘hits’ may tell you what similar proteins in other organisms do, from this you can infer the function of your gene/orf/protein
how do you know if ORF is real or if it’s a pseudogene?
pseudogenes are INACTIVE, 2nd non-functional copy of a gene
DNA –> duplication –> mutation –> classical pseudogene formation (inactivating mutation, deletions that remove promoter or mutations in exons that prevent splicing or introduce stop codons into ORFs)
processed RNA –> cDNA (only coding seq) –> genomic DNA gets inserted into cDNA –> processed pseudogene (can’t be transcribed)
how do you know if ORF is real vs. incorrect prediction?
codon bias may influence DNA seq of ORF
genetic code is degenerate, but not all synonymous codons are used with equal freq –> unequal use of these codons is related to tRNA abundance in diff orgs
expect real ORFs to have codon bias characteristic of other gene IN SAME ORG (YOU SEE CODON BIAS IN CODING REGIONS ONLY)
what is transcriptome?
entire collection of RNA transcripts in a cell
transcriptomics seeks to understand the id and abundance of each RNA in cell
techniques used:
- N. blotting (can only examine 1 mRNA at a time
- microarrays or RNAseq, examine ALL mRNAs in cell at once
what is proteome?
entire collection of proteins that an org can make
proteomics = to understand functional roles of proteins of a species and interplay among proteins
what is the purpose of microarray?
can id all genes that are transcribed
DNA microarray is a slide, spotted with a set of synthetic oligonucleotides, representing all of genes in genome
each of these oligonucleotides seqs corresponds to part of a known gene –> relative location of each spot on slide is known
hybridization experiments with RNA extracted from cells tells you if gene is expressed or not
how do microarrays detect differences in gene expression?
- extract mRNA
- make cDNA reverse transcript, label cDNAs with fluorescent dyes (control = green, experiment = red)
- hybridize to microarray
- laser excitation at dye specific Hz
- detect laser emission
- computer calculates relative levels of hybridized probe
can examine all mRNAs expressed at once
indv oligonuccleoides are needed to detect splice variants
DARK SHADES = higher expression
LIGHT SHADES = lower expression
why is RNA sequencing important?
RNA seq can also be used to assess mRNA expression levels
get mRNAs, convert to cDNA
seq cDNAs using NGS
compare multiple samples
number of reads for a particular mRNA is proportional to its level of expression
green spots represent DOWNREGULATED genes
what is tandem mass spectrometry?
AA seq of protein is revealed
2 spectrometers are used
- first measures the MASS of a given peptide (that was generated from protein digestion)
- second analyzes the peptide after it has been digested into even smaller frags
the proteome is LARGER than the genome (t/f)
true
this larger size is rooted in a number of cell processes
1. alternative splicing
2. RNA editing
3. post-translational covalent mod
what is 2D gel electrophoresis?
used to separate a mix of diff proteins, separation technique that can distinguish hundreds or even thousands of diff proteins in cell extract –> involves 2 diff gel electrophoresis exps (1st = separates by pH/cahrge interactions and 2nd = separates by size)
any given cell of a multicell organism will produce only a subset of proteins in its proteome –> subset that it makes depends on:
- cell type
- stage of development
- environmental conditions
how do you interpret the results of 2D gel electrophoresis?
specific spots may be of special interest
- proteins which are very abundant in cell type
- may be important for that cell’s structure or function
- spots present in only given circumstance
- cells exposed to a hormone versus that are not
- spots present only in abnormal cells
- very common in cancer cells
what is mass spectrometry used for?
to id proteins and measure the MASS-TO-CHARFE RATIO OF IONS
next step of 2D gel is to correlate a given spot with a particular protein
- spot is cut out from gel
- protein is then purified from it
- intact peptide, or products from digestion with trypsin may be analyzed by mass spectrometry
can id post-translational mod because of size increase from original
what can mass spectrometry also id?
protein covalent mods
ex: mass of a phosphorylated protein increases by the mass of a phosphate
what are the steps of proteomic analyses?
first
1. sample
2. digestion
3. machine
4. date collected
5. data analysis
second
1. sample extraction
2. protein mix
3. machine
4. denatured protein data
5. data analysis
what is the Hardy-Weinberg equation?
p^2+2pq+q^2
what does p and q represent in the Hardy-Weinberg equation?
p = dominant allele
q = recessive allele
allele frequencies are given by p and q
genotype frequencies will be given by p^2, 2pq, and q^2
what does it mean when a population is in Hardy-Weinberg equilibrium or not? how do you determine this?
if in Hardy-Weinberg equilibrium = pop IS NOT EVOLVING
if not in H-W equ = pop is EVOLVING and one or more the 5 assumptions is being violated
what are the interpretations from a non-evolving pop?
allele freq are preserved and they are in H-W equilibrium
what are the 3 steps for the Hardy-Weinberg equation?
- calc allele freq from genotype/phenotype freq
- calc expected numbers of each genotype (use the H-W formula to figure out how many homozygotes and heterozygotes you would expect
- compare your expected and observed data
what are the 5 agents of evolutionary change?
- mutation
- gene flow
- non-random mating
- genetic drift
- selection
what is the gene flow H-W equ assumption?
transfer of alleles among pops
EMIgration = transfers alleles OUT of a pop
IMMIgration = transfers alleles IN
what is the assumption of non-random mating?
3 biases in mating
- ASSORTIVE mating –> positive = similar types of mate, negative = opps attract
- ISOLATION by distance –> indvs more apt to mate with neighbor than distantly, located indv, leads to SUBPOPS, proportions of homozygotes larger than predicated by H-W
- INBREEDING –> increases homozygosity, recessive traits are expressed and can result in REDUCTION in vigor and reproductive success (inbreeding depression)
what are the consequences of inbreeding?
results in EXCESS of HOMOZYGOTES compared with random mating and DEFICIENCY of HETEROZYGOTES
what is the inbreeding coefficient F?
closer relations = higher F
sibling matings = riskiest (250x increase at 0.001)
what is the assumption of genetic drift?
H-W assumes INFINITE pop size but real pops are FINITE
allele freqs can change from gen to gen as a result of chance of SAMPLING ERROR when gametes are drawn to form next gen
GENETIC DRIFT = alelle freq change due to CHANCE, affects SMALLER pops more than larger pops
by chance, alleles may be lost from pop –> fixation of particular genotypes in pop
GENETIC DRIFT REDUCES GENETIC VARIABILITY
genetic drifts are WEAKEST in LARGE pops and STRONGEST in small pops
what is the bottleneck theory of genetic drift?
genetic drift often results from pops passing through a pop bottle neck
greater than or equal to consecutive gens when pop size contracts, caused by decrease food supply and increased predation
decrease pop size during bottleneck and increase genetic drift, also increases inbreeding
what is the founder effect?
random sampling of original pop to create a new pop
founder effect is an example of a pop bottleneck
colonists from the mainland colonize an island –> island gene pool is not as variable as the mainland’s
ex: Amish
what is the assumption of no mutations?
H-W assumes no mutation, but mutations do occur
mutations produce genetic variation needed for evolution
mutation alone is NOT A POWERFUL evolunationary force
what is the assumption of no selection?
MUTATION AND SELECTION TOGETHER make a VERY POWERFUL evolutionary force
genotypes (from mutation and recombo) that exhibit differential survival and reproduction = selection
- sexual selection selects for traits that improve mating success
- natural selection selects for traits advantageous for survival
natural selection = survival of the fittest
how does fitness affect allele frequencies?
allele freq CHANGE OVER TIME when diff genotypes have different fitness (pop will evolve)
dynamics differ, depending on whether favored allele is dom or recessive
- if dom allele is favored = rises SHARPLY, hits plateau, slowly approaches fixation (AA=Aa) –> the unfavored recessive will mostly be in heterozygotes where it is HIDDEN FROM SELECTION, difficult to purge unfavored recessive allele completely
- if recessive allele favored = rises SLOWLY at first, aa initially rare (Aa now unfavored, A allele purged from pop)
what are ways of maintaining multiple alleles in gene pool?
- deleterious recessive allele can hide in heretozygote genotype
- there may be heterozygote advantage
- freq-dependent selection
balanced polymorphism/hterezygote superiority = fitness of heretozygote is greater than that of either homozygotes (when there is heterozygote superiority, neither allele can be eliminated by selection)
ex: sickle cell disease with HbS allele
what is freq-dependent selection?
freq-dependent selection occurs when rare alleles have a selective advantage because they are RARE
how do you use H-W to predict potential spread of CCR5-delta32 allele?
CCR5 allele = coreceptor for HIV
some indvs who are resistant to HIV have CCR5 mutaiton
ex: CCR5 delta 32 (frameshift mutation) that introduces a premature stop codon into CCR5 receptor gene, resulting in nonfunctional receptor
homozygotes = immune
you can get WT-WT, WT-delta 32 dimers that are nonfunctional, and heterozygotes that are once infected = more treatable
may increase morbidity
what is the spread of CCR5-delta 32 allele scenario 1?
HIGH HIV infection rate and HIGH initial allele freq of 20%
in human pop with HIGH HIV INFECTION RATE and high. freq of delta 32 allele = delta 32 can spread rapidly (graph will increase freq)
what is the spread of CCR5-delta 32 allele scenario 2?
in areas with LOW HIV infection rates, but HIGH levels of delta-32 –> selection is TOO WEAK to raise delta 32 freq much (graph remains at same freq)
what is the spread of CCR5-delta 32 allele scenario 3?
in sub-Saharan African, HIV infection rate is about 25% but delta 32 allele is almost ABSENT
under these conditions, delta 32 freq will hardly change
what is purifying selection?
natural selection process that removes harmful genetic variations from a pop
