Topic 4 Flashcards
What is the difference in prokaryotes and eukaryotes abou their dna
And where they are
Prokaryotes DNA is inside a nucleoid, less compact
Eukaryotes inside nucleus, very compact
What is special about prokaryotes and eukaryotes chromosome
What I special abiut the prolaryotes
Prokaryotes have a single circular chromosome
Eukaryotes have multiple linear chromosomes
They have a plasmid which is accessory content
What is the ploidy of prokaryotes
Eukaryotes
Haploid
Diploid or haploid
What happens to the gene as organisms get more complex
More complex organism
Highe number of choromosme
More copies of chromosomes
The complexity of the genetic content is increase
What is the genome size definition
Why is it important
The total amount of the genetic content in a haploid organism (one copy of the genome)
So if it’s listed as 3200 for humans, it’s actually the haploid of human not diploid
What is the exception to saying that a more complex organisms has a higher genome size
Doesn’t work all the time Because some organisms of similar complexity have different genome sizes
What is gene density
The average number of genes per mega base (Mb) of genomic DNA
What types of organisms have lower gene density and why
More complex organisms because they have a bigger genes size and more dna between genes (intergenic sequences)
Inverse relationship with gene density
What are intergenic sequences
DNA between genes
When genome size increases in more complex organisms what is actually increasing
More introns
More repetitive DNA
Longer intergenic sequences
NOT JUST GENE NUMBER
What two categories is the human genome split into
Intergenic DNA (> 60% of the content)
Genes and gene related sequences (<40%)
What’s included in intergenic DNA
Other intergenic regions
Genome wide repeats (majority of the genome is repeats)
What is example of Genome wide repeats
Transposons and other mobile DNA’s
What included in Other intergenic regions in intergenic DNA
Unique DNA (regulatory regions, microRNA)
Microsatellites (simple repeats, AC100)
What is genes and gene related sequences split into
Genes (48 Mb)
Related sequences
What is included in related sequences in gene and gene related sequences
Introns and UTR
Gene fragments
Psuedogenes
What are introns and UTRS
UTR are untranslated regions like the poly A tail
Introns include microRNA (non coding functional rna)
What do intergenic sequences encode
DNA sequences that are Transcribed into Functional rna
But not proteins because that RNA doesn’t get translated
What are functional non coding RNA examples
MicroRNA
rRNA
TRNA
snRNA (small nuclear RNA, spliceosome)
Important for cellular function but don’t get translated to proteins
How do you find the percent of genome that’s actually coding
Human genome is 3200 Mb
Genes are 48 Mb
48/3200 = 1.5 % actually coding
Which genes related sequences are non function
Genes fragments
Psudogenes
What are the impotent parts of a chromosome
Kinetochore
Centromeres
Telomeres
Origins of replication
What is the kinetochore
A elaborate protein complex that is on the centromeres to interact with spindle surging segregation in celldvision
Trilaminar: three layered structure
What are the centromeres
How many per chromosome
DNA sequences that are needed for the kinetichore complex to from
This is the first place we see construction of a chromosome
1 centromere per chromosome
What are telomeres and sequence
How many per chromosome
They are TG rich repeats that cal the ends of the chromosome : (TTAGGG)n
Protect from damage and loss
2 pairs per chromosome
What are the origins of replication
Sites on the chromsome where DNA replication machinery assembled and starts replication
Many origins per chromosome
What happens if chromosome has one
No
2
Centromeres
1: equal segregation (one per cell)
None: spindle fibres can’t attach , chromatids can’t move in certain direction (random segregation)
2: chromosomes break, not equal segregation bc of more than one centromere
Why are telomeres so important
They prevent recombination and degradation of DNA (by distinguishing the chromosome ends from the DNA breakage sites)
They act as a specialized origin of replication for replicating the ends of the chromosomes (via telomerase)
What is telomerase
A reverse transcriptase
They reversely replicate the telomeres sequences By using the original telomere sequence at a template
To put in the new chromosome
What are the stages of mitosis
Interphase (G1,S,G2)
Mitosis (M)
In what pint of the cell cycle does dna condense
Between G2 and M
What what pint in the cell cycle does DNA replication happen
Between G1 and S
What parts of the cell cycle mark structural changes of the chromosome
DNA replication
DNA condense
Chromosome segregation (in M phase)
What are the two checkpoints in cell cycle
G1 to S: to check if DNA actually replicated
G2 to M: to check if chromosomes properly aligned before segregation takes place
Interphase is ____
M phase is ____
Replication
Segregation
What is the purpose of the GAP phase (G) in the cell cycle
To prepare for the next phase of the cell cycle
To check for the completion of the previous phase (checkpoints)
What are the key events in S phase
What holds the duplicated chromsomes together
DNA replication starts at the origins and spreads in both directions (many origin sites going up and down)
Each chromosome of a duplicated pair is called a chromatid (both in one pair called sister chromatids)
Cohesion (forms rings to hold them together, important for chromosomal integrity)
What has to happens for the replicates dna to get segregated (metaphase to anaphase)
What did this mean
The cohesin has to be removed through degredation
Protein synthesis and degradation it’s important for mitosis (making and degrading cohesin)
What is bivalent attachement
Both spindles are attached to the kinetochore
This allows proper segregation
What is the MTOC/centosomes
The things that the spindles that attach to the kinetochore on each chromsome come from
In what phase of the cell cycle are the chromosome less compact
When are they most compact
What does thsi mean
Interphase (G1,S,G2)
During mitosis, chromsome condensation allows segregation
From interphase to M PHASE there is a drastic change in chromosome topology
What phase is most important to shape the chromosome structure
Early prophase
Why does dna loop
It does this as a way to package the dna
What are the SMC proteins
Structural maintence of chromosome proteins
Cohesin and condensin
What does condensin do
What happens if degraded
Condenses the looping dna structure to keep it more compact for mitosis
If degraded the dna goes from compact to loose (to go back to g1 phase)
In meiosis 1 and 2 what type of spindle attachment are there and what do they do
Meiosis 1: Monovalent , changes chromosme number from diploid to haploid
Meiosis 2: bivalent, changes chromatin number (sparsrated into 4 cell not two) not chromosome number (because still haploid)
In interphase what are the forms of the chromatin structure (sizes)
In m phase what are the forms of the chromatin structure
Two forms: 10nm (beads on a string) and 30 nm fiber (more compact)
M phase: maximum condensation
Why does dna have to be compact
The diameter of the nucleus is 10-15 micrometers
In a diploid cell the dna is 2 m
So this long dna has to be compacted 1000 to 10,000 times to fit in the nucleus
How is dna compaction achieved
By forming DNA complexes with proteins (ie. chromatin) to from chromosomes
How is a nucleosome formed
The dna is negatively supercoiled into the histone core
What is the advantage of DNA packaging
The histones protect the DNA from damage
Allows for proper segregation during cell division (because stops entanglement)
What is the disadvantage of DNA packaging
the DNA needs to be unwrapped for transcription to happen
So being packages reduces the accessibility of the cellular machinery that’s needs for cell function (repair, recombination, replication,transcription)
What type of structure is a nucleosome
A chromatin structure (dna plus protein)
What are nucleosomes made of
How many times does dna wrap around it
How much commotion happens due to the nucleosome
8 histone protiens with dna wrapped 2x around the histone core
6x
What connects the multiple nucleosomes
The linker dna between nucleosomes
How did they discover the nucleosome
Treated the chromsome with a MNase
Ran gel electrophoresis
Since histone protects DNA from nuclease activity
If digestion is complete (no linker dna) full digestion shows only one 147 bp band
This band is the DNA wrapped around the histone
If incomplete, not just one histone since linker dna not cleaved
So bands at 200 400 600 etc. show since one histone is 200
What is MNase
Micrococcal nuclease
It’s a sequence non specific nucelase
Quickly Cleaves protein free dna but cleaves protein accosiated dna badly
What did the discovery of nucleosome experiment tell us
The nucleosome needs at least 147 bo of dna to form
How long is the linker dna
20-60bp
What amino acids are rich in proteins
Highly basic lysine and arg
Positively charged amino acids
How did the lysine and arg residues affect how we see the histone MW on SDS PAGE
Since positively charged, the lysine and arg make it travel slower on ads page (toward the negative)
This makes the MW seem bigger than we thought
Instead of 10-20 kDa like we thought it shows as 30 kDa
What is special about the n and c term of histone proteins
In between
They have long n term and short c term
In between is the histone fold domain which is made up of alpha helices
What are the diff types of histone subunits that make a histone core for DNA to wrap around (and make nucelosome)
H2A H2B H3 H4
How is a nucleosome assembled
H3 and H4 form a tetramer (since two of each)
H2A and H2B make a heterodimer (only one of each and diff so hetero)
The H3H4 tetramer can bind to the middle and ends of the dna in the inside to make them bent into a circular structure
Then the two of the H2A H2B dimer add onto the tetramer on both sides of the tetramer (so not on the inside)
The amino tails stick outward
In a histone what makes it an octamer
Two dimers of H2A and H2B
One tetramer of H3 H4
Total 8
How does the DNA contact the histones (H3H4)
What is important about this
Hydrogen bonding near the minor grooves of the dna, also the arg lys of histone is postive and the backbone is negative
The contacts are sequence independent (no specific sequence needed to bind to histones)
The hydrogens bonds allow for the energy to being the dna into circle to wrap around
What is special about the n term tails of histones
They are not important for the bending or binding of dna to the histone octamer
They are sensitive to proteases meaning they are not tightly folded in comparison to the histone octamer
They can be modified to regulate the function of the nucleosome
What do the histone tail help with
The tails hide the wrapping of dna to the histone core
And the negative supercoil directionality of the dna
How do the histone tails protrude out
Some tails go between the gaps in the double helices (H3 H2B)
Some go above or below the dna (H4 H2A)
How can the negative supercoiled dna in eukaryotes be relaxed
What is good about being negative supercoil
By topoisomerase
This favours DNA unwinding to allow access to dna during replication transcription and recombination
What are the two topoisomerase sin prokaryotes and what do they do
Gyrase: introduces negative supercoils using atp
Reverse gyrase: keeps genome positive supercoil using atp (used by thermophiles)
What is heterochromatin and euchromatin
What is the staining
Heterochromatin: condensed, dense staining, higher order of nucleosomal dna assembly, closer to nuclear envelope
Euchromatin: open structure, poor staining, less organized nucelosomal dna assembly , centre of nucleus
How did they see the nuclear scaffold/matrix region of the nucleus
They treated the cell with high salt, washed away the proteins
Found there were still proteins in the nucleus that are forming scaffolds close to the nuclear envelope where we see heterochromatin
What causes further condensation of dna onto histones
What does it do and what is special about it
The extra histone: linker histone (H1)
It interacts with the linker DNA to turn the 10nm to 30nm fibre, meaning is also protects the 20-60bp linker region DNA from MNase digestion
Why would we use LYS ARG rich histones for forming chromatin structures
Because they interact tight with negative charged DNA backbone
What forms the 30nm fibre
Nucleosomes plus the H1 linker histone
What are the two form of the 30nm fibre
The solenoid (hollow inside)
Zigzag: the linker region crosses between histones so no hollow inside
If the linker region is short, what form of packaging would the 30 nm fibre take
Solenoid
Longer is zigzag
When the Christin turn to 30 nm. (Getting more compact) what happens
The DNA is less accessible to the DNA dependent enzymes (like polymerases)
This causes less transcription/replication, so less expression
In the 30 nm structure, what is required to stabilize it
The histone tails
They interact with negative DNA on neighbouring nucleosomes since postive charge
So through charge neutralization they allow compaction
How much fold compaction does the 30 nm fibre give
From 6x (bead on string) to 40 x compaction (30nm)
Not enough compaction
How is the rope like structure of the 30 nm fibre made
How big are the loops
Onto a chromosome scaffold there are multiple chromatin loops (40-90kb)
What are the nuclear scaffold proteins that form the 30nm rope loops
Topo II
SMC
What does topo 11 scaffold protein do to form the 30nm loops in a scaffold
Holds the DNA at the base of the loops and makes sure they are topologically isolated from each other
What do SMC scaffold proteins do to form the 30nm loops in a scaffold
Condense and hold the sister chromatids after chromosome duplication into the nuclear scaffold
Provides the foundation for interactions between the nuclear scaffold and the chromosomal DNA
Exaplin the process of forming nuclear scaffold with the 30nm chromatin
How is it undone
During interphase cohesin makes and stabilizes the DNA loops into a more compact structure
Topo 11 comes and bind to the dna to introduce knots by cutting and threading stands through each other (can either make or remove the dna entanglements, right now it’s making them)
The condensin can use dna loop extrusion activity to constrict the the inter and intra molecular interlinks (to untangle them)
What are the three ways during formation of chromatin scaffold that Topo two can make knots
Within one loop
Knots with Nearby loops :
intra (loops from the same dna strand
Inter (loops from diffent dna strands)
What are the histone variants and why are they here
H2A.X
CENP-A
Pack the dna
What is H2A.X
H2A VARIANT, it’s phosphorylated
Its found wherever there’s a double stranded DNA break and is recognized by the DNA repair enzymes
Good indicator of meiosis recombination (which breaks the dna recombine it)
Used for antibodies to recognize the H2A.X to show breakage of DNA that needs repair
What is CENP-A used for
How much similarity does it have to H3
Replaces the H3 , 40-50% similarity to H3
Found in nucleosome that have bound dna with a centromere (found in region of the dna that are centromeric)
Act as a binding site for the kinetochore to allow proper serration and stability
What is used for sperm in mammals packaging
Why
What richness does it have
Sperm nucleus is smaller than somatic nucleus
Use protamines instead of histones, arg rich, packs the dna tightly to for the haploid genome into the sperm nucleus
How could you distinguish between the sperm and somatic dna on a agarose gel restriction digest
See 147 base pairs for somatic dna (full digest)
Sperm cells have no protection from his room since no histones, so you see a smear of a bunch of bands
What do DNA binding proteins (rna pol, dna pol) prefer to interact with
Histone free dna
Want no nucleosome structure
Since dna binding proteins prefers to. Interact with histone free dna what does this mean
The histone octamer hinder dna accessibility
So we need relaxed dna for gene expression
What regulates the accessibility of chromatin
The dynamic nature of the octamer binding to dna
The chromatin remodeling complex changing the binding of histones to the dna to make some regions more open and some more compact
Modification of the N term histone tails
Or all other these this together
What region of the dna on histones is most accessible
The regions closest to the entry and exit points of binding to the histone
Ones just coming into nucleosome structure or just leaving the structure
Not in between because wrapped around the histone
How can chromatin/nucleosome remodelling complex change the movement of the nucleosome
What does it use to do this
NRC (nucleosome remodeling complexes)
Use atp to
Slide the nuceleosomes away from the accessible binding sites
Can eject nucleosome fully out of the region that needs to be transcribed
The histone dimer (H2A H2B) get exchange with other dimer by the remodeling complex. Ex. H2A to H2A.X
How do the remodeling complex use nucleosome positioning to allow dna acceibility
The remodeling proteins bind to the dna and make it so that the sequence between them is less that 147 BP
This causes no nucleosome there since nucleosome need at least 147 BP to form
Makes a nucleosome free region and accessible DNA.
How do the remodeling complex use nucleosome positioning to stop dna acceibility
Bind to the accessible binding site and recruited the histone subunit
makes it so that a nucleosome forms there
Nucelosome prefer to bind what type of DNA
Bent
What nucleotides in the minor groove actually contact the histone and why
AT rich
This is because it’s more flexible (since less h bonding) and can be bent more easily for DNA to bind to histones
Also the electrostatic charge of AT is more negative
Does protamines maintain the nucelosome structure
No
More open dna structure means what in terms of nucleosomes
Less nucelosome formation
How do researches see the position of nuceleosomes in dna
Isolate the nuclei
Treat with MNase to make double stranded DNA breaks between nucelosomes
Treat with a restriction enzyme to make defined end points for all fragments
Run this on agarose gel, then make a replicate on nitrocellulose membrane
Then they design probes for the regions of dna they’re interested in and do southern blot
Found that if the nuceleosomes are randomly placed in the chromsome, cut is done randomly, see a smear of bands
If the nucleosomes are in specific positions next to the sequence they’re interested in, see 160-200 bp bands of the nuceleosomes
When doing the experiment to see nucleosome position, what do they mark the probe bind to
Usually interested in a specific regulatory sequence of the dna
How can the hormone tail be modified
Acetylation
Phosphorylation
Methylation
Ubiquitination
Which amino acid residues get commonly phosphorylated
Serine, tyrosine, threonine
What aA get methylated or acetylated or ubiquinated
Lysine
What does modification of histone tails do
Affect protein association which affects the nucelosome function and gene expression
What is the histone code
The multiple modifications of the histone tail that change the function
What does histone acetylation do
What protein domain does it recruit
HAT adds acetyl group
Reduces positive charge of tail, Opens the dna, Increases gene expression
Recruited the bromodomain
What does histone methylation do
Makes more postive charge, bind tighter to dna, less gene expression
Recruit chromodomain
What proteins interact with unmodified histone tails
Proteins that have the SANT domain
In what way do modifications work together to change gene expression
In a combinatorial way
Combination of these modifications can change chromatin structure and expression
What other way can chromatin structure be regulated
Competition between histone and specific dna binding protiens
What has to happen right after dna replication after S phase
The replicated dna has to be quickly packaged into nucelosome
How do you find if
all old histones are lost and only the new ones are assembled into nucleosomes?
Can get differentially label the histones with radio isotopes
Found that as soon as replication fork forms for replication, the histone start to disassemble from the template strand
The H2A and H2B dimer are released from the original DNA (free floating) and competes with the H3 H4 for binding
But the H3 H4 tetramers stay attached to the dna and randomly segregate to one of the strands (not free floating)
In the end what was the answer to if histone are newly made or reused during replication
Mix of both old and new H3 and H4 / H2A H2B
How are nucleosome modification inherited during replication
Why is this important
The one old histone has a modification, it recruits the enzymes that modify the new histones in the same way
Maintains the epigenetic marks from one generation to the next
What are histone chaperones
What is their charge
Negative charged proteins that complex with histones and allow them to assemble into nucelosomes during replication
What are histone chaperones
Negative charged proteins that complex with histones and allow them to assemble into nucelosomes during replication
How do chaperones actually allow replication
How do chaperones actually assemble nucelosomes
After replication of a region of dna, The histone chaperones are recruited to the ring shaped DNA sliding clamp PCNA (on the DNA)
they dissolve the PCNA right after replication is finished
Then they being and transfer the histone subunits to the newly replicated DNA
How can geneticist see which nucleotide in DNA are modified
Whole genome bisulfite sequencing (WGBS)
The bisulfite only works on unmethylated cytosines
So if the DNA is unmethylated, the bisulfite comes in a removes the amino group (deaminates) the cytosine
This turns the unmethylated cytosine into Uracil.
Compare this new data to the original dna and see that the unchanged cytosines were methylated
How can we see the interactions between genes
HI-C expeiments
What is HI-C sequencing
Get a 3D view of the chromosome interactions in the genome
They cross link the dna using a cross linker (so the dna stays intact)
They lyse the cells, then digest the DNA into small fragments by using Use restriction enzymes to make a sticky end
Need to refill the sticky ends into one end with biotin then close the dna by ligation
Through sonicator they make it into even smaller fragments
They pull down the fragments by the biotin binding to striptoavodine
Then they sequence these fragments
Sequence Tells us the two closely neighboured chromatins that interact with each o the and can see the whole genome architecture of the chromatin structure
